1Fifth Department of Internal Medicine, Tokyo Medical University, Ibaraki-Ken 300-0385; and 2Third Department of Internal Medicine, Nippon Medical School, Tokyo 113-8603, Japan
Submitted 29 December 2003 ; accepted in final form 22 March 2004
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ABSTRACT |
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prostaglandin E2; gastric ulcer; angiogenesis; repair process
It was previously shown (28), in mice, that COX-2 is induced in ulcerated gastric mucosa and that its inhibition by a selective COX-2 inhibitor delays ulcer healing. We have also shown, in the human stomach, that COX-2 is exclusively expressed in gastric mesenchymal cells such as fibroblasts and in inflammatory cells of the ulcer bed and margins (44), suggesting that COX-2 expressed in mesenchymal cells at the ulcer margin plays a key role in the ulcer repair process. However, we have yet to determine the process triggered by COX-2 expression or the role of COX-2-expressing gastric fibroblasts in gastric ulcer healing. Angiogenesis is known to be a critical factor in tissue regeneration (4, 39). A recent study clearly shows that delayed ulcer healing caused by NSAIDs may be due to an imbalance of anti- and proangiogenic factors in serum (24). Thus we considered the possibility that COX-2 may be involved in angiogenesis during gastric ulcer healing by stimulating angiogenic factors in COX-2-expressing mesenchymal cells in the ulcer bed. One such angiogenic factor, VEGF/vascular permeability factor, specifically stimulates endothelial cells (11, 22), and its induction has been shown in COX-2-expressing cancer tissue (23, 43, 45). Therefore, we focused on gastric fibroblasts, which strongly express COX-2 protein immediately beneath necrotic ulcer tissue (44), and examined whether VEGF is released in these gastric fibroblasts via a COX-2-PGE2-dependent autocrine/paracrine pathway.
Thus, to clarify the role of COX-2 in angiogenesis during gastric ulcer healing, we used inflammatory cytokines as tools to induce COX-2 and NS-398, a selective COX-2 inhibitor, as a vehicle to investigate whether COX-2 is involved in VEGF production in human gastric fibroblasts. Finally, we ran immunohistochemical tests to determine the type and location of cells expressing COX-2 and VEGF in human gastric ulcer tissue.
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MATERIALS AND METHODS |
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Human gastric fibroblasts (Hs262.St) were purchased from American Type Culture Collection (Rockville, MD). IL-1 and IL-1
were obtained from R & D (Boston, MA), and NS-398 was kindly provided by Taisho Pharmaceutical (Tokyo, Japan). Mouse anti-human COX-1 antibody (IBL, Gunma, Japan) and mouse anti-human COX-2 antibody (IBL) were used for Western blot analysis. Mouse monoclonal antibodies against vimentin (DAKO, Carpinteria, CA) and COX-2 (Transduction Laboratories, Lexington, KY) were used for immunohistochemical staining (26). A rabbit polyclonal antibody against VEGF (generously provided by Dr. T. Ishiwata, Department of Pathology, Nippon Medical School) was also used for immunohistochemical staining (35).
Preparation of Gastric Fibroblast Supernatant
Gastric fibroblasts (1 x 105) were cultured on 24-well plastic plates in 1 ml of RPMI 1640 medium (Nikken, Tokyo, Japan) supplemented with 10% fetal calf serum and penicillin-streptomycin (GIBCO Life Technologies, Gaithersburg, MD) at 37°C in 5% CO2. Confluent gastric fibroblasts were then washed twice with PBS and cultured in the presence or absence of IL-1 or IL-1
at various concentrations under serum-free condition for 24 h. In some cases, gastric fibroblasts were also cultured in the presence or absence of indomethacin or a selective COX-2 inhibitor, NS-398, at 10 µmol/l. Previous studies (6, 13) have shown that NS-398 at 10 µmol/l selectively inhibits COX-2 activity in vitro. The supernatant was harvested at selected points after stimulation and stored at 20°C. Gastric fibroblasts were also cultured in a similar manner on 6-cm dishes to test COX protein expression by Western blot.
Detection of VEGF and PGE2
The amount of VEGF-165 in culture supernatant was detected with an ELISA kit (IBL). Briefly, a 100-µl aliquot sample was dispensed into a microtiter plate coated with mouse monoclonal antibody against human VEGF-165. After incubation at 37°C for 1 h, the plate was washed with PBS seven times. Anti-human VEGF rabbit IgG Fab'-peroxidase conjugate was then added, and the plate was incubated at 37°C for 30 min and washed nine times with PBS. A 100-µl aliquot of tetramethyl benzidine solution (0.2 mg/ml) was added as substrate and incubated in the absence of light at room temperature for 30 min. The reaction was stopped by addition of 100 µl of 1 N H2SO4. Absorbance at 450 nm was measured by an automatic plate reader. VEGF concentration was determined on a standard curve obtained by a serial dilution of recombinant human VEGF within the range of 01,000 pg/ml. The ELISA kit has been shown to specifically detect VEGF-165 among all isoforms, but 6% cross-reactivity was seen against VEGF-121 (47).
The amount of PGE2 was also detected as described in the instructions of an enzyme immunoassay kit (Assay Design), which included a monoclonal antibody against PGE2 and alkaline phosphatase molecules covalently attached to PGE2.
Western Blot Analysis
COX-1 and COX-2 protein expression in gastric fibroblasts was detected by Western blot analysis as previously described (28). Briefly, cultured fibroblasts were harvested in 25 mM Tris·HCl (pH 8.1) buffer containing 0.25 M sucrose, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 µM pepstatin A, and 1.0 mM EDTA. The pellet was collected by centrifugation at 10,000 g for 2 min and resuspended in the same buffer. CHAPS was added to 1% (wt/vol), and the mixture was stirred for 2 h at 4°C. After centrifugation at 50,000 g for 20 min, the supernatant was loaded onto an anion-exchange column equilibrated with 20 mmol/l Tris·HCl (pH 8.1) and 0.4% CHAPS.
Samples containing 50 µg of protein were separated on 10% acrylamide gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Next, the proteins were electrophoretically transferred to a nitrocellulose membrane and probed with anti-COX-2 antibody specific for human COX-2 protein. Bound antibody was detected with horseradish peroxidase-conjugated antibodies via an enhanced chemiluminescence detection system. The same nitrocellulose membrane was washed as described previously (28) and then reprobed with anti-COX-1 antibody.
Immunofluorescence Histochemistry
Human gastric ulcer tissue samples with perforation were obtained by surgical resection in nine patients (7 men and 2 women, 4479 yr old, mean 60.0 yr old). Tissue was fixed with buffered 10% formalin, embedded in paraffin, and sectioned to a thickness of 3 µm.
Single antibody labeling. The tissue sections were rehydrated and immersed in 0.3% H2O2 in methanol for 30 min to block endogenous peroxidase activity. The sections were incubated with 10% normal horse serum for 30 min to block nonspecific binding of the secondary antibody and then incubated overnight with antivimentin antibody (dilution 1:400) at 4°C. Antibody binding was detected by the avidin-biotin-peroxidase complex method (Vecstatin Elite ABC kit, Vector Laboratories, Burlingame, CA) and visualized using the diaminobenzidine substrate kit (Vector Laboratories) according to the manufacturer's instructions. Nuclei were counterstained with Mayer's hematoxylin.
Negative control immunohistochemical procedures included omission of the primary antibody and replacement of the primary antibody by normal mouse IgG.
Double-labeling procedures. Double labeling using immunofluorescence methods and confocal laser scanning microscopy was used to evaluate the colocalization of immunoreactivity for COX-2 (1:40) and VEGF (1:200). The sections were incubated overnight at 4°C with a mixture of the two primary antibodies. The antibody against COX-2 was reacted with a secondary antibody (horse anti-mouse IgG, dilution 1:100; Vector Laboratories) labeled with FITC. The antibody against VEGF was reacted with a secondary antibody (goat anti-rabbit IgG, dilution 1:100; Vector Laboratories) labeled with Texas red; then nuclear counterstaining was carried out with 4',6-diamidino-2-phenylindole (Sigma, St. Louis, MO) for 15 min to facilitate identification of morphological features.
Immunohistochemical control procedures similar to those described for single labeling were employed in conjunction with dual-staining methods. All preparations were examined with a confocal microscope (model TCS4D/DMIRBE, Leica, Heidelberg, Germany) equipped with argon and argon-krypton laser sources.
Statistical Analysis
Data were analyzed by one-way analysis of variance followed by Fisher's projected least significant difference test as a post hoc test, as appropriate. P < 0.05 was considered statistically significant.
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RESULTS |
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We examined whether human gastric fibroblasts are able to release angiogenic factors when stimulated by inflammatory cytokines such as IL-1, IL-1
, and TNF-
. IL-1
and IL-1
stimulation increased VEGF release from gastric fibroblasts, whereas TNF-
stimulation for 24 h had no effect on VEGF release (Fig. 1A). IL-1
stimulated increases in VEGF release in a dose-dependent manner within a range of 1.2510 ng/ml (Fig. 1B).
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Inflammatory cytokines have been shown to induce COX-2 protein in fibroblasts and macrophages. Therefore, our next step was to examine whether IL-1 induces COX-2 protein production in gastric fibroblasts. Figure 2 shows the result of Western blot analysis with anti-COX-2 antibodies. COX-2 protein expression was remarkably increased by the stimulation of 10 ng/ml IL-1
, even in the presence of 10 µM indomethacin or 10 µM NS-398. We also examined COX-1 protein expression in the same nitrocellulose membrane used above by reprobing with anti-COX-1 antibodies, but expression did not differ significantly among samples (data not shown). The results suggest that IL-1
stimulates COX-2 protein production and VEGF release in gastric fibroblasts within the same dose range. The results also showed that NS-398 and indomethacin had no effect on COX enzyme expression, only affecting enzyme activity.
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Because IL-1 stimulates VEGF release and COX-2 protein production in gastric fibroblasts, we next examined whether IL-1-induced VEGF release is regulated via a COX-dependent pathway. Figure 3 shows that IL-1-stimulated VEGF release was equally inhibited in the presence of 10 µM indomethacin or 10 µM NS-398, although neither inhibitor affected basal VEGF release significantly. We further found that VEGF inhibition by COX inhibitors was restored by the simultaneous addition of PGE2 at 100 µM. These data suggest that PGE2 derived from COX induced by IL-1
stimulation might be involved in VEGF release from gastric fibroblasts.
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To clarify whether gastric fibroblasts can actually release increased amounts of PGE2, we measured PGE2 levels in the supernatant of gastric fibroblasts after IL-1 and IL-1
stimulation. Figure 4 shows that IL-1
and IL-1
induced a significant increase in PGE2 release from gastric fibroblasts at concentrations seen for VEGF production. Because IL-1
and IL-1
did not induce COX-1 protein production in gastric fibroblasts, the increase in PGE2 by the stimulation of these cytokines might be derived from increased COX-2 protein expression. A nonselective COX inhibitor, indomethacin, and a selective COX-2 inhibitor, NS-398, similarly inhibited IL-1
-induced PGE2 production down to control levels, further suggesting that human gastric fibroblasts secrete PGE2 in response to IL-1
via a COX-2-dependent pathway.
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To confirm whether gastric fibroblasts release VEGF in response to PGE2 stimulation, we examined the effect of graded concentrations of PGE2 on VEGF release in fibroblasts. There was a dose-dependent stimulation of VEGF production after treatment with PGE2 at 0.1100 µmol/l. Maximum VEGF release was about three times more than control levels 24 h after PGE2 stimulation (Fig. 5). Because the concentration of PGE2 detected in the supernatant after IL-1 stimulation was
0.11 µmol/l, these results, taken together, suggest that the amount of PGE2 secreted by gastric fibroblasts is enough to induce VEGF release by gastric fibroblasts in an autocrine manner in vitro.
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To clarify the kinetics of IL-1-stimulated VEGF and PGE2 release, we examined IL-1
-induced increases in VEGF and PGE2 release in the course of 24 h of stimulation. After IL-1
stimulation, PGE2 was first detected at 3 h; thereafter, PGE2 levels continued to rise until levels peaked at 24 h. In a similar pattern, VEGF was detected initially 6 h after IL-1
stimulation and continued to increase for 24 h (Fig. 6). The accumulation pattern of VEGF was likely to follow that of PGE2. Taken together, these data imply that human gastric fibroblasts release PGE2 via upregulation of COX-2, and, thereafter, PGE2 stimulates VEGF release in an autocrine or paracrine manner.
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Our previous report showed that COX-2 expression was exclusively localized in gastric mesenchymal cells of the gastric ulcer bed in surgically resected gastric tissue or biopsy specimens of the gastric ulcer margin, as shown immunohistochemically (44). The rate of COX-2-positive cells was also found to significantly increase in the active and healing stages of ulcers, compared with Helicobacter pylori-negative normal gastric mucosa. To analyze the location of VEGF and COX-2 protein in gastric ulcer tissue, we performed double-staining analysis of ulcerated regions by using immunofluorescence-conjugating antibodies.
Numerous inflammatory cells and spindle-shaped cells were found beneath necrotic tissue of the ulcer bed in resected sections (Fig. 7A). Reaction against vimentin was found in all spindle-shaped cells, indicating that these cells are fibroblasts or myofibroblasts (Fig. 7B). COX-2 was expressed in these spindle-shaped mesenchymal cells and also in spherical inflammatory cells of the granulation tissue (Fig. 7C). Strong VEGF immunoreactivity was also observed in the same gastric tissue section of the ulcer bed (Fig. 7D). Double staining of COX-2 and VEGF with immunofluorescence-conjugating antibodies revealed that VEGF and COX-2 were coexpressed in these spindle-shaped and spherical cells of the ulcer bed (Fig. 7, E and F).
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DISCUSSION |
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We previously showed that increased COX-2 expression in gastric tissue of experimentally induced ulcers is involved in the ulcer repair process in mice (28). In addition, we have found strong COX-2 protein induction in fibroblasts and macrophages between necrotic and granulation tissues of the ulcer bed in surgically resected human gastric tissue (44). Nevertheless, we still do not know the role of mesenchymal COX-2 expression in the ulcer repair process in the human stomach. However, our present data raise the possibility that VEGF released from COX-2-expressing fibroblasts might be a key to this repair process, because VEGF has been recently shown to stimulate angiogenesis in the wound repair process: keratinocytes were shown to express VEGF mRNA in the healing process of wounds, and decreased VEGF mRNA expression was shown to impair the healing of skin wounds in genetically diabetic db/db mice (8, 9, 12). Furthermore, in the wound repair process, VEGF responses in vivo might be tied to COX-2 expression, given that celecoxib, a selective COX-2 inhibitor, impaired angiogenesis and delayed gastric ulcer healing, with concomitant decrease in serum VEGF-to-endostatin ratios (24). In addition to our finding in vitro, COX-2-dependent hepatocyte growth factor (HGF) release has also been shown in gastric fibroblasts after IL-1 stimulation (3, 41). HGF is well known as a powerful stimulator of epithelial cell migration and proliferation (42) and, therefore, is considered one of the factors involved in the ulcer repair process. Thus gastric fibroblasts expressing COX-2 at the ulcer margin might play a key role in the ulcer repair process by releasing not only growth factors such as VEGF and HGF, but also PGE2, which has been involved in various defense systems of the gastric mucosa (21, 33).
In the present study, we have further demonstrated that VEGF localized in fibroblast-like spindle cells in surgically resected gastric tissue samples of perforated ulcers. As we previously showed, COX-2 was also found strongly expressed in fibroblasts of the ulcer bed in the human stomach. VEGF has been shown to be colocalized in COX-2-expressing fibroblasts, suggesting that VEGF release from gastric fibroblasts in vivo could also depend on COX-2 expression, as seen in our isolated cultured gastric fibroblasts. Our in vivo immunohistochemical colocalization of COX-2 and VEGF in gastric fibroblasts of the ulcer bed is consistent with other in vivo studies showing a relationship between COX-2 expression and angiogenesis. In chronic and proliferating granuloma of the rat, COX-2 mRNA has been shown to increase with neovascularization in parallel with VEGF mRNA (25). Indomethacin and SC-236, a selective COX-2 inhibitor, have been shown to inhibit neovascularization in a dose-dependent manner in a mouse corneal model of angiogenesis (26). In carrageenin-induced granulation tissue in rats, COX-2-derived PGE2 has been shown to play a significant role in angiogenesis through VEGF formation (16). However, these in vivo studies have failed to show where COX-2 and VEGF expressions are localized. In the present study, we have clearly shown that COX-2 and VEGF are expressed in mesenchymal cells, such as fibroblasts of the ulcer bed, and might interact as we have shown in vitro.
VEGF-induced angiogenesis has been mostly shown in connection with cancer development. One recent study clearly showed that colon cancer cells release a variety of COX-2-dependent proangiogenic growth factors, whereas endothelial cell migration and tubular formation were observed only in COX-2-expressing cancer cells cocultured with endothelial cells (45). In a recent study, we also observed the immunohistochemical colocalization of VEGF and COX-2 in cancer cells of human gastric carcinoma (43). Many studies in addition to ours show VEGF expression in cancer cells (15, 29). Here, however, we show COX-2 and VEGF localized mainly in mesenchymal cells scattered in inflammatory granulation tissue of the ulcer bed. Only faint COX-2 expression can be observed in regenerated epithelial cells of the ulcer margin, as we previously showed (44). Recently, another study (1) also showed that COX-2 and VEGF are expressed in fibroblasts of granulation tissue induced by sponge implants in rats. Therefore, although COX-2 might play important roles in VEGF expression in cancer tissue and inflammatory granulation, the expression site for this key factor might differ in each tissue.
Regarding NSAID-caused gastric mucosal injury, COX-2, in addition to COX-1, has been found to be a target enzyme for NSAIDs (46). Conventional NSAIDs inhibiting COX-1 and COX-2 have been shown to cause serious and significant ulcer complications, including bleeding and perforation (7, 37). Although it is clear that selective COX-2 inhibitors such as celecoxib and rofecoxib cause less gastric damage than conventional NSAIDs (18, 38), these selective COX-2 inhibitors have still been shown to occasionally cause serious gastrointestinal complications (17). Although we do not know whether gastric fibroblasts express COX-2 in NSAID-induced gastric ulcers, if COX-2 were expressed, its biological activity would be inhibited. Thus we must consider the possibility that NSAID-induced inhibition of the COX-2-dependent ulcer repair process might ultimately lead to the serious complications of gastric mucosal damage attributed to NSAIDs.
In conclusion, COX-2 plays a key role in regulating VEGF production in gastric fibroblasts. Gastric fibroblasts strongly expressing COX-2 and VEGF at the ulcer margin might be involved in the ulcer repair process.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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