Gastric Fibroblasts Research Articles

Cyclooxygenase-2-regulated vascular endothelial growth factor release in gastric fibroblasts.

VEGF is a highly specific stimulator of endothelial cells and may play an important role in angiogenesis in the process of tissue regeneration. We previously showed that cyclooxygenase-2 (COX-2) expressed in mesenchymal cells of the ulcer bed is involved in the ulcer repair process. To clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we investigated the relation between COX-2 expression and VEGF production in human gastric fibroblasts in vivo and in vitro. Gastric fibroblasts were cultured in RPMI 1640 with and without IL-1alpha or IL-1beta in the presence or absence of NS-398, a selective COX-2 inhibitor. Supernatant VEGF and PGE(2) concentrations were measured by enzyme-linked immunosorbent assay. COX-2 expression in fibroblasts was determined by Western blot analysis. VEGF and COX-2 expression in surgical resections of human gastric ulcer tissue was examined immunohistochemically. IL-1 dose dependently enhanced VEGF release in cultured gastric fibroblasts after a 24-h stimulation. IL-1 also stimulated PGE(2) production in gastric fibroblasts via COX-2 induction. NS-398 significantly suppressed VEGF and PGE(2) release from IL-1-stimulated gastric fibroblasts; concurrent addition of PGE(2) restored NS-398-inhibited VEGF release. COX-2 and VEGF immunoreactivity were colocalized in fibroblast-like cells in the ulcer bed of gastric tissues. These results suggest that COX-2 plays a key role in VEGF production in gastric fibroblasts stimulated by IL-1 in vitro and that angiogenesis induced by the COX-2-VEGF pathway might be involved in gastric ulcer healing.

Expression of urokinase-type plasminogen activator and its receptor in gastric fibroblasts and effects of nonsteroidal antiinflammatory drugs and prostaglandin.

In acute inflammatory condition, little is known about the expression of the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) in the gastric fibroblasts. To clarify the role of human gastric fibroblasts in acute inflammatory conditions such as gastric ulcer, the effects of interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha on the expression of uPA and uPAR, which were suggested to be associated with tissue remodeling, in gastric fibroblasts were investigated. The expression level of uPA mRNA and the amount of uPA antigen increased significantly on treatment with each concentration of IL-1beta (1 and 10 ng/ml) and 10 ng/ml TNF-alpha. On the other hand, the amounts of uPA antigen on cell surfaces were not affected significantly by IL-1beta and TNF-alpha stimulation. The expression level of uPAR mRNA increased in a dose-dependent manner on IL-1beta stimulation. The effect of indomethacin on uPA and uPAR expression in these cells was also examined. When gastric fibroblasts were treated with 50 microM indomethacin, the expression level of uPA mRNA decreased significantly, and the amount of uPA antigen in the culture medium and on cell surfaces decreased significantly with indomethacin in a dose-dependent manner. The increased uPAR mRNA expression caused by IL-1beta was reduced to the basal level by treatment with 50 microM indomethacin. Furthermore, we investigated the role of prostaglandin E2 (PGE2), which is suggested to play major roles in acute inflammation of the stomatch, on uPA and uPAR expression in gastric fibroblasts. The expression level of uPAR mRNA and the amount of uPA antigen on cell surfaces increased in a dose-dependent manner on treatment with PGE2 (10 and 50 ng/ml). These results suggest that uPA and uPAR expression in gastric fibroblasts is involved in the regulating system of PGE2 and that NSAIDs may delay healing of gastric mucosal injury in part through suppressing uPA production via inhibition of endogenous PG production.

Gastric restitution is inhibited by dexamethasone, which is reversed by hepatocyte growth factor and rebamipide.

Glucocorticoids have been shown to induce peptic ulcers, especially when co-administered with NSAIDs. Hepatocyte growth factor (HGF) plays a role in gastric ulcer repair, facilitating the restitution of gastric mucosal epithelial cells. HGF expression is induced by PGs in gastric fibroblasts. We hypothesized that dexamethasone (DEX) may inhibit PG production and HGF expression, thus inhibiting HGF-induced gastric epithelial restitution. To investigate the effect of DEX on gastric restitution, using cultured gastric cells, the role of HGF in the restitution inhibited by DEX, and the effect of rebamipide on DEX- inhibited restitution. Human gastric fibroblasts were prepared from human stomach obtained at surgery; PGE2 and HGF is determined by ELISA; Restitution was assessed by the round wound restitution model, using coculture of gastric fibroblasts and epithelial cells; COX-2 and HGF mRNA were quantified by TaqMan RT-PCR system. 1. DEX inhibited HGF mRNA and COX-2 mRNA. Accordingly, it inhibited PGE2 and HGF release. 2. DEX inhibited the restitution of gastric cells. 3. The inhibition of restitution was reversed by HGF and rebamipide to the same extent. 4. Rebamipide induced PGE2 and HGF. DEX inhibits restitution via HGF depletion, and rebamipide reverses the inhibited restitution by HGF induction.

Heregulin released from gastric fibroblasts via a COX-2-PGE2 pathway stimulates ErbB3 tyrosine phosphorylation in MKN-28 gastric epithelial cells

Heregulin released from gastric fibroblasts via a COX-2-PGE2 pathway stimulates ErbB3 tyrosine phosphorylation in MKN-28 gastric epithelial cells

Induction of urokinase-type plasminogen activator and its receptor in the gastric fibroblasts and effects of nonsteroidal anti-inflammatory drugs and prostaglandin

Induction of urokinase-type plasminogen activator and its receptor in the gastric fibroblasts and effects of nonsteroidal anti-inflammatory drugs and prostaglandin

BFGF stimulates VEGF production through MEK-, PI-3 kinase- and COX-2-PGE2-dependent pathway in gastric fibroblasts

bFGF stimulates VEGF production through MEK-, PI-3 kinase- and COX-2-PGE2-dependent pathway in gastric fibroblasts

Transformation of fibroblast to myofibroblast by application of Hp on the gastric surface epithelial cell using two-layered culture of gastric mucous cell and fibroblast: Its relation to leptin and rebamipide

Transformation of fibroblast to myofibroblast by application of Hp on the gastric surface epithelial cell using two-layered culture of gastric mucous cell and fibroblast: Its relation to leptin and rebamipide

Lasp-1 binds to non-muscle F-actin in vitro and is localized within multiple sites of dynamic actin assembly in vivo.

Lasp-1 has been identified as a signaling molecule that is phosphorylated upon elevation of [cAMP]i in pancreas, intestine and gastric mucosa and is selectively expressed in cells within epithelial tissues. In the gastric parietal cell, cAMP-dependent phosphorylation induces the partial translocation of lasp-1 to the apically directed F-actin-rich canalicular membrane, which is the site of active HCl secretion. Lasp-1 is an unusual modular protein that contains an N-terminal LIM domain, a C-terminal SH3 domain and two internal nebulin repeats. Domain-based analyses have recently categorized this protein as an epithelial representative of the nebulin family, which also includes the actin binding, muscle-specific proteins, nebulin, nebulette and N-RAP. In this study, we show that lasp-1 binds to non-muscle filamentous (F) actin in vitro in a phosphorylation-dependent manner. In addition, we provide evidence that lasp-1 is concentrated within focal complexes as well as in the leading edges of lamellipodia and the tips of filopodia in non-transformed gastric fibroblasts. In actin pull-down assays, the apparent K(d) of bacterially expressed his-tagged lasp-1 binding to F-actin was 2 micro M with a saturation stoichiometry of approximately 1:7. Phosphorylation of recombinant lasp-1 with recombinant PKA increased the K(d) and decreased the B(max) for lasp-1 binding to F-actin. Microsequencing and site-directed mutagenesis localized the major in vivo and in vitro PKA-dependent phosphorylation sites in rabbit lasp-1 to S(99) and S(146). BLAST searches confirmed that both sites are conserved in human and chicken homologues. Transfection of lasp-1 cDNA encoding for alanine substitutions at S(99) and S(146), into parietal cells appeared to suppress the cAMP-dependent translocation of lasp-1 to the intracellular canalicular region. In gastric fibroblasts, exposure to the protein kinase C activator, PMA, was correlated with the translocation of lasp-1 into newly formed F-actin-rich lamellipodial extensions and nascent focal complexes. Since lasp-1 does not appear to be phosphorylated by PKC, these data suggest that other mechanisms in addition to cAMP-dependent phosphorylation can mediate the translocation of lasp-1 to regions of dynamic actin turnover. The localization of lasp-1 to these subcellular regions under a range of experimental conditions and the phosphorylation-dependent regulation of this protein in F-actin rich epithelial cells suggests an integral and possibly cell-specific role in modulating cytoskeletal/membrane-based cellular activities.

Study on the increment of the production of gastric mucus in rats treated with Opuntia ficus indica (L.) Mill. cladodes

Opuntia ficus indica cladodes are used in traditional medicine of many countries for their cicatrisant activity. The major components of cladodes are carbohydrate-containing polymers, which consist of a mixture of mucilage and pectin. In this paper we studied the cytoprotective effects of cladodes on experimental ethanol-induced ulcer in rat. The O. ficus indica cladodes administration gives rise to cytoprotection phenomena by breaking up the epithelial cells and stimulating an increase in mucus production. When O. ficus indica cladodes are administered as a preventive therapy, keep the gastric mucosa under normal condition by preventing mucus dissolution caused by ethanol and favouring mucus production. An increase of mucus production is also observed during the course of the curative treatment. The treatment with O. ficus indica cladodes provokes an increase in the number of secretory cells. Probably, the gastric fibroblasts are involved in the antiulcer activity.

Role of nuclear factor-kappaB in gastric ulcer healing in rats.

We investigated the role of nuclear factor-kappaB (NF-kappaB) in gastric ulcer healing in rats. NF-kappaB was activated in ulcerated tissue but not in normal mucosa, and the level of the activation was decreased with ulcer healing. NF-kappaB activation was observed in fibroblasts, monocytes/macrophages, and neutrophils. Treatment of gastric fibroblasts, isolated from the ulcer base, with interleukin-1beta activated NF-kappaB and the subsequently induced cyclooxygenase-2 and cytokine-induced neutrophil chemoattractant-1 (CINC-1) mRNA expression. Inhibition of activated NF-kappaB action resulted in suppression of both their mRNA expression and increases in PGE(2) and CINC-1 levels induced by interleukin-1beta. Persistent prevention of NF-kappaB activation caused an impairment of ulcer healing in rats. Gene expression of interleukin-1beta, CINC-1, cyclooxygenase-2, and inducible nitric oxide synthase in ulcerated tissue had been inhibited before the delay in ulcer healing became manifest. The increased levels of cyclooxygenase-2 protein and PGE(2) production were also reduced. These results demonstrate that NF-kappaB, activated in ulcerated tissue, might upregulate the expression of healing-promoting factors responsible for gastric ulcer healing in rats.

The LIM and SH3 domain-containing protein, lasp-1, may link the cAMP signaling pathway with dynamic membrane restructuring activities in ion transporting epithelia.

Lasp-1 is a unique LIM and src homology 3 (SH3) domain-containing protein that was initially identified as a 40 kDa cAMP-dependent phosphoprotein in the HCl-secreting gastric parietal cell. Because cAMP is a potent stimulator of parietal cell acid secretion, we have hypothesized that changes in lasp-1 phosphorylation might be involved in the regulation of ion transport-related activities, perhaps by modulating interactions among cytoskeletal and/or vesicle-associated proteins. In this study, we demonstrate that the cAMP-dependent acid secretory agonist, histamine, induces a rapid, sustained rise in parietal cell lasp-1 phosphorylation and this increase in phosphorylation is closely correlated with the acid secretory response. In addition, elevation of intracellular cAMP concentrations appear to induce a partial redistribution of lasp-1 from the cell cortex, where it predominates along with the gamma-isoform of actin in unstimulated cells, to the beta-actin enriched, apically-directed intracellular canalicular region, which is the site of active proton transport in the parietal cell. Additional studies demonstrate that although lasp-1 mRNA and protein are expressed in a wide range of tissues, the expression is specific for certain actin-rich cell types present within these tissues. For example, gastric chief cells, which contain relatively little F-actin and secrete the enzyme, pepsinogen, by regulated exocytosis, do not appear to express lasp-1. Similarly, lasp-1 was not detected in pancreatic acinar cells, which secrete enzymes by similar mechanisms and also contain relatively low levels of F-actin. Lasp-1 also was not detectable in proximal tubules in the kidney, in gastrointestinal smooth muscle, heart or skeletal muscle. In contrast, expression was prominent in the cortical regions of ion-transporting duct cells in the pancreas and in the salivary parotid gland as well as in certain F-actin-rich cells in the distal tubule/collecting duct. Interestingly, moderate levels of expression were also detected in podocytes present in renal glomeruli and in vascular endothelium. In primary cultures of gastric fibroblasts, lasp-1 was present mainly within the tips of lamellipodia and at the leading edges of membrane ruffles. Taken together these results support the hypothesis that the lasp-1 plays an important role in the regulation of dynamic actin-based, cytoskeletal activities. Agonist-dependent changes in lasp-1 phosphorylation may also serve to regulate actin-associated ion transport activities, not only in the parietal cell but also in certain other F-actin-rich secretory epithelial cell types.

Cyclooxygenase-2 (COX-2) regulates vascular endothelial-growth factor (VEGF) production by gastric fibroblasts stimulated with interleukin-1(IL-1) and transforming growth factor-b (TGF-M)

Cyclooxygenase-2 (COX-2) regulates vascular endothelial-growth factor (VEGF) production by gastric fibroblasts stimulated with interleukin-1(IL-1) and transforming growth factor-b (TGF-M)

The effect of NSAIDs and a COX-2 specific inhibitor on Helicobacter pylori-induced PGE2 and HGF in human gastric fibroblasts.

There is compelling evidence for the pivotal role of Helicobacter pylori in the pathogenesis of gastrointestinal ulcer disease. However, despite the bacterium's toxicity, the majority of H. pylori infections are not accompanied by gastric ulcers. This implies the existence of a host mechanism offsetting H. pylori toxicity. To evaluate gastric fibroblasts' expression of hepatocyte growth factor (HGF), which is known to facilitate gastric ulcer healing, in the presence of H. pylori; to compare the effect on H. pylori-induced HGF expression of a COX-2 selective inhibitor with that of nonselective nonsteroidal anti-inflammatory drugs Human gastric fibroblasts were cultured from human gastric mucosa obtained at surgery. Prostaglandin E2 (PGE2) and HGF were measured by EIA. The expression of COX-2 mRNA was assessed by the TaqMan quantitative RT-PCR system. H. pylori increased PGE2 release in gastric fibroblasts. H. pylori induced expression of COX-2 mRNA, which indicates that PG induction by H. pylori is through COX-2. Sulindac sulphide, etodolac and NS 398 all inhibited H. pylori-induced PGE2 release to the same extent. These agents also inhibited H. pylori-induced HGF release. Gastric fibroblasts produce PG and HGF in response to the presence of H. pylori, which may be considered part of the human body's defensive reaction to H. pylori toxicity. This defensive mechanism is inhibited not only by COX-2 nonselective NSAIDs but also by a COX-2 selective inhibitor. These findings indicate the importance of COX-2 in chronic H. pylori infection.

TGF-beta1 secreted by gastric fibroblasts up-regulates CD44H expression and stimulates the peritoneal metastatic ability of scirrhous gastric cancer cells.

Gastric fibroblast-derived conditioned medium and TGF-beta1 stimulated the adhesion ability of scirrhous gastric cancer cells to mesothelial cells, but not that of well-differentiated gastric cancer cells. The CD44H expression of scirrhous gastric cancer cells was significantly up-regulated by gastric fibroblast-derived CM and TGF-beta1. The stimulated adhesion ability and CD44H expression was significantly inhibited by anti TGF-beta1 neutralizing antibody. These findings suggested that TGF-beta1 secreted by gastric fibroblasts up-regulated the CD44H expression of scirrhous gastric cancer cells, which resulted in the stimulation of the adhesion ability of scirrhous gastric cancer cells to the mesothelium, and increased the peritoneal dissemination potential. These results might explain one of the reasons why scirrhous gastric carcinomas develop frequent peritoneal dissemination.

Expression of heregulin α, erbB2, and erbB3 and their influences on proliferation of gastric epithelial cells

Background & Aims: Heregulins (HRGs) regulate the proliferation and differentiation of various cell types. However, very little is known about their function in the gastrointestinal tract. The aim of this study was to investigate the role of HRGs on gastrointestinal cells. Methods: We examined the expression of erbB receptors and HRG-α in human gastric cancer cell lines, rat gastrointestinal epithelial cells, and human gastric fibroblasts by Western blot analysis or reverse-transcription polymerase chain reaction. Receptor phosphorylation and heterodimerization induced by HRG-α were detected by Western blot analysis. We also evaluated the in vitro effects of HRG-α on cell proliferation and restitution. Results: Cancer cell lines and rat epithelial cells expressed erbB2 and erbB3, but protein expression of erbB4 was not detected. HRG-α was detected only in gastric fibroblasts. HRG-α activated tyrosine phosphorylation of epidermal growth factor receptor (EGFR), erbB2, and erbB3 and induced not only erbB3/erbB2 but also erbB3/EGFR and erbB2/EGFR heterodimer formation in MKN-28 cancer cells. Simultaneous cultivation of MKN-28 cells with gastric fibroblasts resulted in tyrosine phosphorylation of erbB3 in MKN-28 cells. HRG-α also stimulated proliferation of MKN-28 cells and gastric epithelial cells. Conclusions: The data suggest that HRG-α may affect epithelial cell proliferation through mesenchymal-epithelial interaction in the gastric mucosa. GASTROENTEROLOGY 1999;117:1119-1127

Tranilast and cisplatin as an experimental combination therapy for scirrhous gastric cancer.

We examined the effect of Tranilast on the reduction of the administered dose of cisplatin using a scirrhous gastric cancer model. Scirrhous gastric cancer cell line, OCUM-2M, and gastric fibroblasts, NF-10, were used. The IC50 values of CDDP to OCUM-2M cells were decreased by Tranilast in vitro. The combination treatment with Tranilast and CDDP decreased the xenografted tumor size. The combination therapy decreased fibrosis and mitosis, and increased apoptosis. These findings suggest that Tranilast increased the CDDP response on scirrhous gastric cancer. The combination treatment with Tranilast and CDDP may be useful as a new therapy for scirrhous gastric carcinoma.

Nonsteroidal Anti-inflammatory Drugs May Delay the Repair of Gastric Mucosa by Suppressing Prostaglandin-Mediated Increase of Hepatocyte Growth Factor Production

Prostaglandins (PGs), hepatocyte growth factor (HGF), and induction of cyclooxygenase (PG synthetase, COX) play important roles in the repair process of gastric mucosa. We hypothesized that nonsteroidal anti-inflammatory drugs (NSAIDs), including indomethacin (IND), retard the healing of ulcers by suppressing these factors. In this study, we investigated the effects of cytokines, growth factors, and IND on production of PG and HGF, and induction of COX using cultured human gastric fibroblasts. Exogenous PGs significantly increased HGF production in a dose-dependent manner. Among various potential stimulants tested, interleukin-1β (IL-1β) dramatically increased PGE2production and significantly stimulated HGF production. IL-1β induced COX-2 but not COX-1 protein. IND significantly reduced both basal and IL-1β-induced PGE2release and HGF production. These results suggest that the IL-1β — PG — HGF pathway plays a role in the repair process of gastric mucosa. Further, NSAIDs may delay the healing of gastric mucosal ulcer, in part through suppression of HGF expression via inhibition of endogenous PG production.

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.

In vitro wound repair by human gastric fibroblasts: implications for ulcer healing.

Fibroblasts modulate epithelial biological activities and play a key role in the ulcer healing process. There is no information regarding the biological response of human gastric fibroblasts to regulatory compounds. The aim of this study was to assess the effects of growth factors and prostaglandins on an in vitro model of human gastric fibroblast wound repair. Subconfluent fibroblast cultures were used to study proliferative responses, determined by [3H]thymidine incorporation into DNA. In vitro wound repair was determined in confluent fibroblast monolayers after mechanical denudation. The presence of putative growth factors secreted by fibroblasts was studied in conditioned medium by heparin-affinity chromatography and immunodetection with specific antibodies. Serum and platelet-derived growth factor (PDGF) -BB induced a dramatic increase in both gastric fibroblast proliferation and closure of wounded cell monolayers, whereas these activities were inhibited by both transforming growth factor (TGF) -beta1 and prostaglandin E1. Basal activities in unstimulated gastric fibroblasts were lower than those obtained in skin fibroblasts. Conditioned medium stimulated fibroblast proliferation and wound repair activity, which was inhibited by the addition of suramin, and was partially dependent on the presence of PDGF-like factor. PDGF is a major, autocrine promotor of human gastric fibroblast-dependent wound repair activities, which are inhibited by prostaglandins and TGF-beta. These findings might be important for future therapeutic ulcer healing approaches.

Gastric epithelial cells secrete a PDGF-like peptide, a potent mitogen for human gastric fibroblasts.

To investigate whether gastric epithelial cells secrete growth factors involved in stromal cell growth, we examined the effects of conditioned media obtained from gastric cancer cells on murine BALB/c 3T3 cells and primary cultured human gastric fibroblasts. Conditioned media from MKN-1 gastric cancer cells were applied to a heparin-affinity column. The fraction eluted from the column at 0.4 M NaCl stimulated DNA synthesis and phosphorylation of PDGF alpha-receptors on tyrosine in BALB/c 3T3 cells. The fraction-induced stimulation of DNA synthesis in gastric fibroblasts was more marked than in BALB/c 3T3 cells. However, the fraction failed to stimulate DNA synthesis in CHO-ER cells overexpressing EGF receptors and phosphorylation of PDGF beta-receptors on tyrosine in BALB/c 3T3 cells. Immunoblot analysis of the media confirmed that PDGF-AA-like peptides are released from gastric cancer cells, immortalized gastric epithelial cells, and primary cultured gastric epithelial cells. Anti-PDGF neutralizing antibodies produced only a partial inhibition of 0.4 M NaCl fraction-induced enhanced DNA synthesis. Thus, in addition to PDGF-AA peptide, other bioactive substance(s) are probably released from MKN-1 gastric cancer cells. Our results suggest that gastric epithelial cells secrete PDGF-AA-like peptides responsible for stromal cell growth through paracrine mechanisms.