Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.     

Cyclooxygenase-2 selective and nitric oxide-releasing nonsteroidal anti-inflammatory drugs and gastric mucosal responses.

Occurrence of gastrointestinal damage and delayed healing of pre-existing ulcer are commonly observed in association with clinical use of nonsteroidal antiinflammatory drugs (NSAIDs). We examined the effects of NS-398, the cyclooxygenase (COX)-2 selective inhibitor, and nitric oxide (NO)- releasing aspirin (NCX-4016) on gastric mucosal ulcerogenic and healing responses in experimental animals, in comparison with those of nonselective COX inhibitors such as indomethacin and aspirin. Indomethacin and aspirin given orally were ulcerogenic by themselves in rat stomachs, while either NS-398 or NCX-4016 was not ulcerogenic at the doses which exert the equipotent antiinflammatory action with indomethacin or aspirin. Among these NSAIDs, only NCX-4016 showed a dose-dependent protection against gastric lesions induced by HCl/ethanol in rats. On the other hand, the healing of gastric ulcers induced in mice by thermal-cauterization was significantly delayed by repeated administration of these NSAIDs for more than 7 days, except NCX-4016. Gastric mucosal prostaglandin contents were reduced by indomethacin, aspirin and NCX-4016 in both normal and ulcerated mucosa, while NS-398 significantly decreased prostaglandin generation only in the ulcerated mucosa. Oral administration of NCX-4016 in pylorus-ligated rats and mice increased the levels of NO metabolites in the gastric contents. In addition, both NS-398 and NCX-4016 showed an equipotent anti-inflammatory effect against carrageenan-induced paw edema in rats as compared with indomethacin and aspirin. These results suggest that both indomethacin and aspirin are ulcerogenic by themselves and impair the healing of pre-existing gastric ulcers as well. The former action is due to inhibition of COX-1, while the latter effect may be accounted for by inhibition of COX-2 and mimicked by NS-398, the COX-2 selective NSAID. NCX-4016, despite inhibiting both COX-1 and COX-2, protects the stomach against damage and preserves the healing response of gastric ulcers, probably because of the beneficial action of NO.     

Implication of gastrin in cyclooxygenase-2 expression in Helicobacter pylori infected gastric ulceration

Gastroduodenal ulcerations have worldwide distribution and the infection with Helicobacter pylori (HP) has been implicated in pathogenesis of this disease. The HP infection is usually accompanied by hypergastrinemia and enhanced generation of prostaglandins (PG), both implicated in the pathogenesis of peptic ulcerations but no study has been undertaken to assess the relationship between the HP infection and coexpression of gastrin and cyclooxygenases (COX), the rate limiting enzymes in the PG production. Since HP infection, usually accompanying peptic ulcerations, results in increased release of gastrin, a potent gastric mitogen that might be capable to induce COX-2 and to generate PG, we decided 1) to compare the seroprevalence of HP and its cytotoxic protein, CagA, in gastric ulcer patients with those in age- and gender-matched controls; 2) to determine the gene expression of gastrin and its receptors (CCK(B)-R) at the margin of gastric ulcer and in the mucosa of antrum and corpus before and after successful eradication of HP, 3) to assess the plasma levels and gastric luminal contents of gastrin before and after HP eradication and 4) to examine the mRNA and enzyme protein expression of COX-1 and COX-2 as well as the PGE2 generation in ulcer margin tissue and gastric antral and fundic mucosa before and after the HP eradication. The trial material included 20 patients with gastric ulcer and 40 age- and gender-matched controls. Anti-HP and anti-CagA IgG seroprevalence was estimated by specific antisera using ELISA tests. Gene expressions of gastrin, CCK(B)-R, COX-1 and COX-2 were examined using RT-PCR with beta-actin as a reference and employing Western blotting for COX-2 expression, while gastrin and PGE2 were measured by RIA. All gastric ulcers were located at smaller curvature within the antral mucosal area. The seroprevalence of HP, especially that expressing CagA, was significantly higher in gastric ulcers (85%) than in controls (62.5%). Both gastrin and CCK(B)-R mRNA were detected by RT-PCR in ulcer margin and gastrin mRNA was overexpressed in remaining antral mucosa, while CCK(B)-R mRNA was overexpressed in fundic mucosa of HP infected patients. Similarly, COX-2 mRNA and protein were found in margin of gastric ulcer and in the HP infected antral and fundic mucosa but not in the mucosa of HP eradicated patients in whom ulcers completely healed and gastrin was expressed only in antrum, CCK(B)-R only in corpus, while COX-1 was detected both in antrum and corpus. HP positive gastric ulcer patients showed about three times higher levels of plasma immunoreactive gastrin and about 50% higher luminal gastrin contents than the HP negative controls and this increased plasma and luminal gastrin was normalized following the HP eradication. A significant fall in gastrin and CCK(B)-R mRNA expression was noticed six weeks after HP eradication in gastric antral and fundic mucosa, while COX-2 mRNA completely disappeared after this treatment. We conclude that 1) HP infected gastric ulcer margin coexpresses gastrin, its receptors (CCK(B)-R), and COX-2; 2) HP infection may be implicated in gastric ulceration via increased release of gastrin that could be responsible for the overexpression of COX-2 that in turn could help ulcer healing through the stimulation of mucosal cell growth, restoration of the glandular structure and angiogenesis in the ulcer area and 3) gastrin produced in HP infected antral mucosa seems to be involved in the induction of COX-2 and PG production by this enzyme and this may contribute to the ulcer healing.     

Role of cyclooxygenase isoforms in gastric mucosal defence.

A complex system of interacting mediators exists in the gastric mucosa to strengthen its resistance against injury. In this system prostaglandins play an important role. Prostaglandin biosynthesis is catalysed by the enzyme cyclooxygenase (COX), which exists in two isoforms, COX-1 and COX-2. Initially the concept was developed that COX-1 functions as housekeeping enzyme, whereas COX-2 yields prostaglandins involved in pathophysiological reactions such as inflammation. In the gastrointestinal tract, the maintenance of mucosal integrity was attributed exclusively to COX-1 without a contribution of COX-2 and ulcerogenic effects of non-steroidal anti-inflammatory drugs (NSAIDs) were believed to be the consequence of inhibition of COX-1. Recent findings, however, indicate that both COX-1 and COX-2 either alone or in concert contribute to gastric mucosal defence. Thus, in normal rat gastric mucosa specific inhibition of COX-1 does not elicit mucosal lesions despite near-maximal suppression of gastric prostaglandin formation. When a selective COX-2 inhibitor which is not ulcerogenic when given alone is added to the COX-1 inhibitor, severe gastric damage develops. In contrast to normal gastric mucosa which requires simultaneous inhibition of COX-1 and COX-2 for breakdown of mucosal resistance, in the acid-challenged rat stomach inhibition of COX-1 alone results in dose-dependent injury which is further increased by additional inhibition of COX-2 enzyme activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. COX-2 inhibitors do not damage the normal or acid-challenged gastric mucosa when given alone. However, when nitric oxide formation is suppressed or afferent nerves are defunctionalized, specific inhibition of COX-2 induces severe gastric damage. Ischemia-reperfusion of the gastric artery is associated with up-regulation of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone augment ischemia-reperfusion-induced gastric damage up to four-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE(2). Selective inhibition of COX-1 is less effective. Furthermore, COX-2 inhibitors antagonize the protective effect of a mild irritant or intragastric peptone perfusion in the rat stomach, whereas the protection induced by chronic administration of endotoxin is mediated by COX-1. Finally, an important function of COX-2 is the acceleration of ulcer healing. COX-2 is up-regulated in chronic gastric ulcers and inhibitors of COX-2 impair the healing of ulcers to the same extent as non-selective NSAIDs. Taken together, these observations show that both COX isoenzymes are essential factors in mucosal defence with specific contributions in various physiological and pathophysiological situations.     

Role of cyclooxygenase-2 in gastric mucosal defense.

Two isoenzymes of cyclooxygenase (COX), the key enzyme in prostaglandin (PG) biosynthesis, COX-1 and COX-2, have been identified. COX-1 was proposed to regulate physiological functions, COX-2 to mediate pathophysiological reactions such as inflammation. In particular, it was suggested that maintenance of gastric mucosal integrity relies exclusively on COX-1. Recently, it was shown that a selective COX-1 inhibitor does not damage the mucosa in the healthy rat stomach, although mucosal prostaglandin formation is near-maximally suppressed. However, concurrent treatment with a COX-1 and a COX-2 inhibitor induces severe gastric damage. This indicates that in normal mucosa both COX-1 and COX-2 have to be inhibited to evoke ulcerogenic effects. In the acid-challenged rat stomach inhibition of COX-1 alone is associated with dose-dependent injury which is aggravated by additional inhibition of COX-2 activity or prevention of acid-induced up-regulation of COX-2 expression by dexamethasone. After acid exposure, COX-2 inhibitors cause substantial gastric injury when nitric oxide formation is suppressed or afferent nerves are defunctionalized. Ischemia-reperfusion of the gastric artery increases levels of COX-2 but not COX-1 mRNA. COX-2 inhibitors or dexamethasone aggravate ischemia-reperfusion-induced mucosal damage up to 4-fold, an effect abolished by concurrent administration of 16,16-dimethyl-PGE2. Furthermore, the protective effects elicited by a mild irritant or intragastric peptone perfusion are antagonized by COX-2 inhibitors. Finally, COX-2 expression is increased in experimental ulcers. COX-2 inhibitors delay the healing of chronic gastric ulcers in experimental animals and decrease epithelial cell proliferation, angiogenesis and maturation of the granulation tissue to the same extent as non-steroidal anti-inflammatory drugs. These observations indicate that, in contrast to the initial concept, COX-2 plays an important role in gastric mucosal defense.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Healing of chronic gastric ulcers in diabetic rats treated with native aspirin, nitric oxide (NO)-derivative of aspirin and cyclooxygenase (COX)-2 inhibitor.

Previous studies have demonstrated that the gastric mucosa of diabetic rats is highly vulnerable to acute injury but the influence of nonsteroidal anti-inflammatory drugs (NSAID) and their new nitric oxide (NO) releasing derivatives of aspirin (NO-ASA) on the ulcer healing under diabetic conditions has been little studied. In this study streptozocin (STZ, 70 mg/kg injected intraperitoneally) was used to induce diabetes mellitus in rats. Four weeks after STZ injection, gastric ulcers were induced using the acetic acid method and rats with gastric ulcers received the treatment with 1) aspirin (ASA, 30 mg/kg-d i.g.), 2) NO-ASA applied in equimolar dose of 50 mg/kg-d i.g., 3) rofecoxib (5 mg/kg-d i.g.), the selective cyclooxygenase-(COX)-2 inhibitor and 4) SNAP (5 mg/kg-d i.g.), a donor of NO, combined with ASA (30 mg/kg-d i.g.). Ten days after the induction of the ulcers, the healing rate and the gastric blood flow (GBF) were measured by planimetry and hydrogen (H(2))-gas clearance method, respectively and the plasma cytokine such as IL-1beta, TNF-alpha and IL-10 were determined. In addition, the effect of insulin (4 IU/day/rat i.p.) with or without the blockade of NO-synthase by L-NNA (20 mg/kg-d i.p.) on the ulcer healing and the GBF in non-diabetic and diabetic rats was determined. In the diabetic rats, a significant delay in ulcer healing (approximately by 300%) was observed with an accompanied decrease in the GBF at ulcer margin. The prolongation of the healing in diabetic animals was associated with an increase in the plasma cytokine (IL-1beta, TNF-alpha and IL-10) levels. ASA and rofecoxib, that significantly suppressed the mucosal prostaglandin (PG) E(2) generation in ulcer area, delayed significantly the rate of ulcer healing and decreased the GBF at ulcer margin, while elevating plasma IL-1beta, TNF-alpha and IL-10 concentrations in non-diabetic rats and these alterations were significantly augmented in diabetic animals. In contrast to ASA, the treatment with NO-ASA failed to influence both, the ulcer healing and GBF at ulcer margin and significantly attenuated the plasma levels of IL-1beta, TNF-alpha and IL-10 as compared to those recorded in ASA- or rofecoxib-treated animals. Co-treatment of SNAP with native ASA abolished the deleterious effect of ASA on ulcer healing, GBF at ulcer margin and luminal NO release in diabetic rats. Administration of insulin in rats with diabetes, opposed the delay in ulcer healing, and the fall in the GBF at ulcer margin and these effects were counteracted by the concurrent treatment with L-NNA. We conclude that: 1) ulcer healing is dramatically impaired in experimental diabetes and this effect involves the fall in the gastric microcirculation at the ulcer margin and increased release of proinflammatory cytokines; 2) classic NSAID such as ASA and selective COX-2 inhibitors such as rofecoxib, prolong ulcer healing under diabetic conditions probably due to suppression of endogenous PG and the fall in the GBF at the ulcer margin suggesting that both COX isoforms, namely, COX-1 and COX-2, are important sources of PG during ulcer healing in diabetes; and 3) NO-ASA counteracts the impairment of ulcer healing in diabetic rats induced by ASA, mainly due to the release of NO that compensates for PG deficiency resulting in enhancement in the GBF at ulcer margin and suppression of cytokine release in the ulcer area.     

Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation

Because neurotensin (NT) and its high-affinity receptor (NTR1) modulate immune responses, chloride secretion, and epithelial cell proliferation, we sought to investigate their role in the repair process that follows the development of mucosal injuries during a persistent inflammation. Colonic NT and NTR1, mRNA, and protein significantly increased only after dextran sodium sulfate (DSS)-induced inflammatory damage developed. Colitis-induced body weight loss, colonic myeloperoxidase activity, and histological damage were significantly enhanced by SR-48642 administration, a nonpeptide NTR1 antagonist, whereas continuous NT infusion ameliorated colitis outcome. To evaluate the NT and NTR1 role in tissue healing, mucosal inflammatory injury was established administering 3% DSS for 5 days. After DSS discontinuation, mice rapidly gained weight, ulcers were healed, and colonic NT, NTR1, and cyclooxygenase (COX)-2 mRNA levels were upregulated, whereas SR-48642 treatment caused a further body weight loss, ulcer enlargement, and a blunted colonic COX-2 mRNA upregulation. In a wound-healing model in vitro, NT-induced cell migration in the denuded area was inhibited by indomethacin but not by an antitransforming growth factor-beta neutralizing antibody. Furthermore, NT significantly increased COX-2 mRNA levels by 2.4-fold and stimulated PGE(2) release in HT-29 cells. These findings suggest that NT and NTR1 are part of the network activated after mucosal injuries and that NT stimulates epithelial restitution at least, in part, through a COX-2 dependent pathway.     

Cyclooxygenase-2/prostaglandin E2 accelerates the healing of gastric ulcers via EP4 receptors

We examined the involvement of cyclooxygenase (COX)-1 as well as COX-2 in the healing of gastric ulcers and investigated which prostaglandin (PG) EP receptor subtype is responsible for the healing-promoting action of PGE2. Male SD rats and C57BL/6 mice, including wild-type, COX-1(-/-), and COX-2(-/-), were used. Gastric ulcers were produced by thermocauterization under ether anesthesia. Gastric ulcer healing was significantly delayed in both rats and mice by indomethacin and rofecoxib but not SC-560 given for 14 days after ulceration. The impaired healing was also observed in COX-2(-/-) but not COX-1(-/-) mice. Mucosal PGE2 content increased after ulceration, and this response was significantly suppressed by indomethacin and rofecoxib but not SC-560. The delayed healing in mice caused by indomethacin was significantly reversed by the coadministration of 11-deoxy-PGE1 (EP3/EP4 agonist) but not other prostanoids, including the EP1, EP2, and EP3 agonists. By contrast, CJ-42794 (selective EP(4) antagonist) significantly delayed the ulcer healing in rats and mice. VEGF expression and angiogenesis were both upregulated in the ulcerated mucosa, and these responses were suppressed by indomethacin, rofocoxib, and CJ-42794. The expression of VEGF in primary rat gastric fibroblasts was increased by PGE2 or AE1-329 (EP4 agonist), and these responses were both attenuated by coadministration of CJ-42794. These results confirmed the importance of COX-2/PGE2 in the healing mechanism of gastric ulcers and further suggested that the healing-promoting action of PGE2 is mediated by the activation of EP4 receptors and is associated with VEGF expression.     

Inhibition of both COX-1 and COX-2 is required for development of gastric damage in response to nonsteroidal antiinflammatory drugs.

We examined the gastric ulcerogenic property of selective COX-1 and/or COX-2 inhibitors in rats, and investigated whether COX-1 inhibition is by itself sufficient for induction of gastric damage. Animals fasted for 18 h were given various COX inhibitors p.o., either alone or in combination, and they were killed 8 h later. The nonselective COX inhibitors such as indomethacin, naproxen and dicrofenac inhibited PG production, increased gastric motility, and provoked severe gastric lesions. In contrast, the selective COX-2 inhibitor rofecoxib did not induce any damage in the stomach, with no effect on the mucosal PGE(2) contents and gastric motility. Likewise, the selective COX-1 inhibitor SC-560 also did not cause gastric damage, despite causing a significant decrease in PGE(2) contents. The combined administration of SC-560 and rofecoxib, however, provoked gross damage in the gastric mucosa, in a dose-dependent manner. SC-560 also caused a marked gastric hypermotility, whereas rofecoxib had no effect on basal gastric motor activity. On the other hand, the COX-2 mRNA was expressed in the stomach after administration of SC-560, while the normal gastric mucosa expressed only COX-1 mRNA but not COX-2 mRNA. These results suggest that the gastric ulcerogenic property of conventional NSAIDs is not accounted for solely by COX-1 inhibition and requires the inhibition of both COX-1 and COX-2. The inhibition of COX-1 up-regulates the COX-2 expression, and this may counteract the deleterious influences, such as gastric hypermotility and the subsequent events, due to a PG deficiency caused by COX-1 inhibition.     

Role of capsaicin sensory nerves and EGF in the healing of gastric ulcer in rats

Accumulating evidence indicates that capsaicin sensitive afferent fibers play a pivotal role not only in gastroprotection but also in ulcer healing. Denervation of capsaicin sensitive afferent fibers exerts an adverse action on these effects. However, whether such an action is mediated through a depression on epidermal growth factor (EGF) is undefined. In this study, the effects of denervation of sensory neurons with capsaicin (100 mg/kg, s.c.) on acetic acid-induced chronic gastric ulcers and their relationship with the EGF expression in salivary glands, serum and gastric mucosa were investigated. Capsaicin significantly increased ulcer size, decreased gastric mucosal cell proliferation at the ulcer margin, angiogenesis in the granulation tissue and also gastric mucus content. Ulcer induction by itself dramatically elevated EGF levels in salivary glands and serum on day 1 and 4, and also in the gastric mucosa on day 4. However, capsaicin completely abolished these effects. It is concluded that stimulation of EGF expression in salivary glands and serum may be one of the mechanisms by which capsaicin sensitive nerves contribute to the gastroprotective and ulcer healing actions in the stomach.     

Annexin-1 modulates repair of gastric mucosal injury

Annexin-1 is a glucocorticoid-inducible protein that plays an important effector role in the resolution of inflammation and has recently been shown to contribute to the resistance of the stomach to injury. Using an integrated genetic and pharmacological approach, we have tested the hypothesis that annexin-1 contributes to the healing of mucosal injury, given that such injury is accompanied by an inflammatory response, which is often associated with an overexpression of annexin-1 expression. Gastric ulcers were induced in mice through serosal application of acetic acid. Annexin-1 expression during the healing of the ulcers was examined. The effects on gastric ulcer healing of treatment with an annexin-1 mimetic (Ac2-26), an antagonist of the annexin-1 receptor (Boc2), or a glucocorticoid (dexamethasone) were examined. Finally, susceptibility to and healing of indomethacin-induced gastric lesions were compared in wild-type and annexin-1-deficient mice. Expression of annexin-1 was significantly increased in the gastric ulcer margin throughout the healing process. Treatment with an annexin-1 mimetic (Ac2-26) significantly enhanced gastric ulcer healing. In contrast, both dexamethasone and an formyl peptide receptor-like-1 (FPRL-1) antagonist impaired the early phase of ulcer healing. Annexin-1-deficient mice exhibited the same susceptibility as wild-type mice to indomethacin-induced gastric damage, but the healing of that damage was impaired in the former. These data support the hypothesis that annexin-1 contributes significantly to the process of healing of gastric mucosal damage.     

Evaluation of Acetic Acid-Induced Chronic Gastric Ulcer Healing by Propionyl-L-Carnitine Administration

The aim of our study was to investigate the healing effect of propionyl-L-carnitine (PLC) on chronic gastric ulcers and its underlying mechanisms. This study included rats with gastric ulcers induced by applying serosal glacial acetic acid. These rats were then given either saline (vehicle) or PLC at doses of 60 and 120 mg/kg, administered orally 3 days after ulcer induction for 14 consecutive days. Our study found that treatment with PLC resulted in a reduction of the gastric ulcer area, a faster rate of ulcer healing, and stimulated mucosal restoration. Additionally, the treatment with PLC reduced the number of Iba-1+ M1 macrophages while increasing the number of galectin-3+ M2 macrophages, as well as desmin+ microvessels, and α-SMA+ myofibroblasts in the gastric ulcer bed. The mRNA expression of COX-2, eNOS, TGF-β1, VEGFA, and EGF in the ulcerated gastric mucosa was greater in the PLC-treated groups compared with the vehicle-treated rats. In conclusion, these findings suggest that PLC treatment may accelerate gastric ulcer healing by stimulating mucosal reconstruction, macrophage polarization, angiogenesis, and fibroblast proliferation, as well as fibroblast-myofibroblast transition. This process is associated with the upregulation of TGF-β1, VEGFA, and EGF, as well as modulation of the cyclooxygenase/nitric oxide synthase systems.     

A vascular endothelial growth factor mimetic accelerates gastric ulcer healing in an iNOS-dependent manner

Angiogenesis is crucial to all types of wound healing, including gastric ulcer healing. The most potent promoter of angiogenesis is vascular endothelial growth factor (VEGF). We hypothesized that a 15-amino acid peptide designed to mimic the angiogenic action of VEGF would accelerate gastric ulcer healing. Gastric ulcers were induced in mice by serosal application of acetic acid. Treatment with the VEGF mimetic accelerated gastric ulcer healing when administered orally or intraperitoneally, at a dose of 50 ng/kg or greater. Such healing was not observed when the reverse sequence pentadecapeptide or the full-length VEGF protein was administered. Contrary to our hypothesis, the VEGF mimetic did not significantly increase angiogenesis in the ulcerated stomach. The enhancement of ulcer healing by the VEGF mimetic occurred independently of cyclooxygenase-2 (COX-2) activity but was blocked by inhibitors of inducible nitric oxide synthase (iNOS). These results demonstrate that a VEGF mimetic is a potent stimulus for gastric ulcer healing, even when given orally. The effects of the mimetic were independent of stimulatory effects on angiogenesis and COX-2 activity but were dependent on iNOS-derived NO production.     

Relationship between vascular endothelial growth factor and angiogenesis in spontaneous and indomethacin-delayed healing of acetic acid-induced gastric ulcers in rats.

Angiogenesis is an important event for gastric ulcer healing. Vascular endothelial growth factor (VEGF) is known to be a potent stimulator of angiogenesis. This study consequently examined VEGF production, VEGF mRNA expression and angiogenesis during the spontaneous and indomethacin-delayed healing of acetic acid-induced ulcers in rats. The production of VEGF, taking place in the normal mucosa, was significantly elevated by ulceration. The mRNA expression of three isoforms of VEGF (VEGF188, VEGF164 and VEGF120) was also detected. Following the increase in VEGF production, angiogenesis was significantly promoted in the ulcer base. VEGF-immunoreactivity was observed in granulocytes, fibroblasts and regenerated epithelial cells. Indomethacin markedly inhibited prostaglandin E2 synthesis in the ulcer base, resulting in the prevention of ulcer healing. Angiogenesis was also significantly inhibited by indomethacin, but neither VEGF production nor VEGF mRNA expression was reduced. Such results suggest that VEGF might play a role in angiogenesis in the spontaneous healing of gastric ulcers in rats. However, the inhibition of angiogenesis in indomethacin-delayed ulcer healing is not explainable on VEGF expression.     

Heregulin-alpha and heregulin-beta expression is linked to a COX-2-PGE2 pathway in human gastric fibroblasts

We have previously shown heregulin (HRG)-alpha expression in human gastric fibroblasts and its stimulation of gastric epithelial cell growth. Although cyclooxygenase (COX)-2 has also been shown to stimulate growth factor production in these cells, the interaction between COX-2 and HRG remains unknown. Conditioned media (CM) from gastric fibroblasts incubated with PGE(2) or interleukin (IL)-1beta, a well known COX-2 inducer, were analyzed for their effect on erbB3 tyrosine phosphorylation in MKN28 gastric epithelial cells. HRG protein expression in fibroblast lysates and CM was also examined by western blot. HRG-alpha and HRG-beta mRNA expression in gastric fibroblasts and human gastric tissue was examined by real-time quantitative PCR. HRG and COX-2 expressions in surgical resections of human gastric ulcer tissue were examined immunohistochemically. CM from fibroblasts incubated with PGE(2), or IL-1beta, stimulated erbB3 phosphorylation in MKN28 cells. Preincubation of the fibroblasts with celecoxib, a selective COX-2 inhibitor, suppressed CM-induced erbB3 phosphorylation. This inhibition was reversed by exogenous PGE(2). As with erbB3 phophorylation, IL-1beta stimulated both HRG-alpha and HRG-beta mRNA expression, as well as HRG release into gastric fibroblast CM. IL-1beta-stimulated HRG expression and release were also inhibited by celecoxib, and exogenous PGE(2) restored this inhibitory effect, suggesting the activation of an IL-1beta-COX-2-PGE(2) pathway that culminates in the release of HRG from fibroblasts. HRG-alpha and HRG-beta mRNA levels were significantly higher in gastric ulcer tissue than in normal gastric mucosa. HRG immunoreactivity was found in interstitial cells of the gastric ulcer bed and coexpressed with COX-2. These results suggest that HRG might be a new member of the growth factor family involved in the COX-2-dependent ulcer repair process.     

Bacterial colonization and healing of gastric ulcers: the effects of epidermal growth factor

Experimental gastric ulcers are rapidly colonized by various bacteria, resulting in significantly impaired healing. Epidermal growth factor (EGF) is capable of preventing bacterial colonization of the healthy intestinal mucosa. In this study, we examined the possibility that EGF accelerates gastric ulcer healing by reducing bacterial colonization of the ulcer. Gastric ulcers were induced by serosal application of acetic acid. The effect of daily administration of EGF on ulcer healing and bacterial colonization was assessed and compared with the effect of daily treatment with broad-spectrum antibiotics. EGF administration reduced colonization levels and accelerated ulcer healing as effectively as the antibiotic treatment. EGF was without effect on acid secretion or neutrophil infiltration into the ulcer. Bacterial growth was not inhibited in the presence of EGF in vitro. These results demonstrate that EGF reduces bacterial colonization during an established infection of a compromised mucosal surface. This effect may contribute to the ability of EGF to accelerate gastric ulcer healing. This effect is acid independent and not due to an anti-inflammatory effect or to direct bactericidal actions.