Dynamics of changes in vascular endothelial growth factor (VEGF) expression and angiogenesis in stress-induced gastric ulceration in rats.

Angiogenesis and VEGF play a major role in many repair processes such as healing of gastric ulceration.The present study was undertaken to assess the dynamics of changes in VEGF expression and angiogenesis in stress-induced gastric ulcers in rats. Acute gastric ulceration was induced using a water-immersion and restraint stress method. The VEGF expression, angiogenesis, size of area and depth of ulcers in gastric specimens were evaluated. The study shows that as early as one day after the development of ulcers there is a significant increase in both the expression of the VEGF protein and the number of newly formed microvessels, while an abrupt decrease in VEGF expression, observed on the fifth day, results in a decreased intensity of angiogenesis. Moreover, it has been demonstrated that the increase in VEGF expression and angiogenesis is accompanied by a reduction in the size of area and depth of stress-induced ulcers in rats. Six days after ulcer development both VEGF expression and angiogenesis return to normal levels.     

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.     

Endothelial nitric oxide synthase modulates gastric ulcer healing in rats

Nitric oxide has been shown to be beneficial for gastric ulcer healing. We determined the relative effects of endothelial and inducible nitric oxide synthases on gastric ulcer healing in rats. Ulcers were induced by serosal application of acetic acid. Ulcer severity, angiogenesis, and nitric oxide synthase expression were assessed 3-10 days later. The effects of inhibitors of nitric oxide synthase were also examined. Inducible nitric oxide synthase mRNA was only detected in ulcerated tissue (maximal at day 3), whereas the endothelial isoform mRNA was detected in normal tissue and increased during ulcer healing. Inducible nitric oxide synthase was expressed in inflammatory cells in the ulcer bed, whereas endothelial nitric oxide synthase was found in the vascular endothelium and in some mucosal cells in both normal and ulcerated tissues. Angiogenesis changed in parallel with endothelial nitric oxide synthase expression. N(6)-(iminoethyl)-L-lysine did not affect angiogenesis or ulcer healing, while N(G)-nitro-L-arginine methyl ester significantly reduced both. In conclusion, endothelial nitric oxide synthase, but not the inducible isoform, plays a significant role in gastric ulcer healing.     

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.     

Role of p38 mitogen-activated protein kinase in the healing of gastric ulcers in rats.

p38 belongs to the mitogen-activated protein kinase family and plays a crucial role in cellular responses to both cytokines and various stresses. We investigated the role of p38 in the healing of experimental gastric ulcers. Gastric ulcers were induced by submucosal injection of acetic acid solution into male rats. Western blotting and a kinase assay examined the p38 activity and phosphorylation state in ulcerated tisue. After orally administering FR167653 (p38 kinase inhibitor) for 3 to 14 days, the production level of cytokines and the protein-level expression of cyclooxygenase and inducible nitric oxide synthase were examined by enzyme-linked immunosorbent assay and Western blotting. Only in fibroblasts and macrophages/monocytes in ulcerated tissue, p38 was found to be phosphorylated with an elevated kinase activity level. FR 167653 inhibited the activity of p38, yet had no effect on its phosphorylation state. The drug significantly impaired ulcer healing (without affecting acid secretion) and angiogenesis in the ulcer base. The production of interleukin-1beta and tumor necrosis factor-alpha were significantly reduced after FR167653 treatment. In addition the expression of cyclooxygenase-2 and inducible nitric oxide synthase proteins increased PGE2 generation and NOx secretion in the ulcerated stomach were suppressed by FR167653. From these findings, we conclude that p38, activated by gastric ulceration, might play some role in the healing of gastic ulcers in rats.     

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.     

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.     

Effect of local injection with basic fibroblast growth factor (BFGF) and neutralizing antibody to BFGF on gastric ulcer healing, gastric secretion, angiogenesis and gastric blood flow.

Exogenous administration of bFGF was shown to accelerate tissue repair predominantly due to an increase in the formation of new microvessels (angiogenesis) suggesting that bFGF plays an important role in healing of gastric ulcer. This study was designed: 1) to examine the effect of local application of bFGF with or without neutralizing antibody (NA) to bFGF and 2) to determine the role of gastric secretion, gastric blood flow (GBF) at the ulcer margin and angiogenesis during gastric ulcer healing with or without local application of NA, bFGF or the combination of NA and bFGF. Chronic gastric ulcers were induced in Wistar rats by subserosal application of acetic acid (ulcer area 28 mm2) and gastric secretion during ulcer healing was assessed using animals additionally equipped with chronic gastric fistulas. The bFGF without or with NA to bFGF (10 ng/100 microl]), irrelevant antibodies (rabbit IgG; 10 microg/100 microl) or vehicle (saline) were locally injected into the subserosa immediately upon ulcer induction (day 0) and at day 2. Rats with acetic acid ulcers without subserosal injections served as controls. At day 11, all animals were anaesthetized and GBF was determined at the ulcer base, ulcer margin as well as in intact mucosa using the H2-gas clearance technique and the area of gastric ulcers was measured by planimetry. Gastric mucosa with ulcer was excised and the percentage of area covered with blood vessels, the number of fibroblasts and the percentage of connective tissue at the ulcer edge was assessed by histology. The gastric ulcers were healed spontaneously in control vehicle-treated rats at day 11 and this was accompanied by the significant increase in the GBF and number of microvessels in the ulcer area. The gastric secretion was suppressed immediately after ulcer induction and increased significantly at day 2 and day 11 but failed to return to that recorded in intact animals. In contrast, local application of bFGF inhibited gastric acid and pepsin outputs at each study time intervals tested and this effect was reversed by addition of NA to bFGF. Locally applied bFGF accelerated significantly ulcer healing and this was accompanied by the greater rise in the GBF of ulcer margin and more marked increase in number of microvessels as compared to those in vehicle-treated rats. Subserosal application of NA to bFGF prolonged significantly the ulcer healing and this effect was accompanied by a significant fall in the GBF at the ulcer margin and a decrease in number of capillaries in ulcer bed without significant alteration in gastric acid and pepsin outputs. The ulcer healing effect of bFGF and accompanying increase in the GBF at ulcer margin and in thenumber of microvessels as well as inhibition of gastric acid secretion evoked by bFGF were significantly attenuated by the addition of NA to bFGF. The number of fibroblasts and the distribution of connective tissue did not differ between groups studied. We conclude that; 1) depletion of endogenous bFGF at the ulcer area by specific NA to bFGF delays healing of gastric ulcers, reduces angiogenesis of ulcer bed and impairs the microcirculatory effect of this growth factor at the ulcer margin indicating that the availability of bFGF in the ulcer area plays a crucial role in the ulcer healing through induction of angiogenesis; 2) this prominent antiulcer effect of locally applied bFGF depends, at least in part, upon the inhibition of acid secretion by this peptide.     

Involvement of heat shock proteins in the healing of acetic acid-induced gastric ulcers in rats.

The present study examined the expression of 73-kDa of heat shock cognate protein (HSC70), 72-kDa of heat shock protein (HSP70) and 47-kDa of HSP (HSP47) observed in the ulcer healing process in rats. Gastric ulcers were induced by a luminal application of acetic acid in male Donryu rats. During the ulcer healing process, the expression of HSPs in the ulcerated tissue was determined. A high level of HSC70 expression was observed both in the normal mucosa and ulcerated tissue, but the level did not change upon ulceration and ulcer healing. While HSP70 and HSP47 were markedly expressed in the ulcer base during ulceration, and decreased with ulcer healing. HSP70 expression in the ulcer margin was gradually increased with ulcer healing. Omeprazole accelerated the healing of gastric ulcers with strong inhibition of gastric acid secretion, while indomethactin delayed in ulcer healing despite slight inhibition of gastric acid secretion. Omperazole enhanced the expression of HSP70 both in the ulcer margin and base, but it reduced HSP47 expression in the ulcer base Indomethacin markedly enhanced HSP47 expression only in the ulcer base. In conclusion, the expression of HSP70 and HSP47 is changed during ulcer healing. Furthermore, it was suggested that the enhanced expression of HSP70 is involved in acceleration of ulcer healing, but overexpression of HSP47 is involved in delayed ulcer healing.     

The Probiotic Mixture VSL#3 Accelerates Gastric Ulcer Healing by Stimulating Vascular Endothelial Growth Factor

Studies assessing the effect and mechanism of probiotics on diseases of the upper gastrointestinal tract (GI) including gastric ulcers are limited despite extensive work and promising results of this therapeutic option for other GI diseases. In this study, we investigated the mechanisms by which the probiotic mixture VSL#3 (a mixture of eight probiotic bacteria including Lactobacilli, Bifidobacteria and Streptococcus species) heals acetic acid induced gastric ulcer in rats. VSL#3 was administered orally at low (6×10⁹ bacteria) or high (1.2×10¹⁰ bacteria) dosages from day 3 after ulcer induction for 14 consecutive days. VSL#3 treatments significantly enhanced gastric ulcer healing in a dose-dependent manner. To assess the mechanism(s) whereby VSL#3 exerted its protective effects, we quantified the gene expression of several pro-inflammatory cytokines, protein and expression of stomach mucin-Muc5ac, regulatory cytokine-IL-10, COX-2 and various growth factors. Of all the components examined, only expression and protein production of VEGF was increased 332-fold on day 7 in the ulcerated tissues of animals treated with VSL#3. Predictably, animals treated with VEGF neutralizing antibody significantly delayed gastric ulcer healing in VSL#3 treated animals. This is the first report to demonstrate high efficacy of the probiotic mixture VSL#3 in enhancing gastric ulcer healing. Probiotic efficacy was effective at higher concentrations of VSL#3 by specifically increasing the expression and production of angiogenesis promoting growth factors, primarily VEGF.     

Characterization of "unhealed gastric ulcers" produced with chronic exposure of acetic acid ulcers to indomethacin in rats.

We previously discovered that a 4-wk course of indomethacin delivered to rats with acetic acid ulcers resulted in production of "unhealed gastric ulcers" that persisted for up to 12 wks after treatment cessation. The present study examined the mechanism underlying such "unhealed gastric ulcers" with biochemical and histological procedures. "Unhealed gastric ulcers" were induced with a 4-wk indomethacin treatment (1 mg/kg, twice daily) in rats with acetic acid ulcers. Two and 4 wks after treatment cessation, ulcer size was significantly larger in rats receiving indomethacin compared with control animals. Ulcerated tissue prostaglandin E2 levels were significantly lower during indomethacin treatment, but the levels tended to increase after treatment cessation compared with levels measure in the group receiving vehicle. Myeloperoxidase activity levels were significantly higher during indomethacin treatment; such levels persisted after treatment cessation. Histologically, greater degrees of fibrosis and neutrophil accumulation, as well as a lesser degree of angiogenesis were observed in the "unhealed gastric ulcers" compared to ulcers that healed in a normal fashion. It was concluded that severe fibrosis, persistent neutrophil infiltration, and poor angiogenesis in the ulcer base might represent factors involved in the mechanism underlying production of "unhealed gastric ulcers".     

Reduction of EGF is associated with the delay of ulcer healing by cigarette smoking

Cigarette smoking is associated with peptic ulcer diseases. Smokers have lower levels of salivary epidermal growth factor (EGF) than nonsmokers. We investigated whether reduction of EGF is involved in the delay of gastric ulcer healing by cigarette smoking. Rats with acetic acid-induced ulcers were exposed to cigarette smoke (0, 2, or 4% vol/vol) 1 day after ulcer induction. EGF level was elevated 1 day after ulcer induction in salivary glands and serum, and 4 days after ulcer induction in the gastric mucosa. However, cigarette smoke depressed these beneficial effects and EGF mRNA expression in salivary glands and gastric mucosa. Cigarette smoke delayed gastric ulcer healing and reduced cell proliferation, angiogenesis, and mucus synthesis. Exogenous EGF (10 and 20 microg/kg i.v.) before smoke exposure reversed the adverse effects of cigarette smoke, whereas vascular endothelial growth factor level and nitric oxide synthase activity were unaffected. It is concluded that the detrimental effect of cigarette smoke on ulcer healing is a consequence of reduction of angiogenesis, cell proliferation, and mucus secretion through the depressive action on EGF biosynthesis and its mRNA expression in salivary glands and gastric mucosa.     

Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.     

Endothelin-1, an ulcer inducer, promotes gastric ulcer healing via mobilizing gastric myofibroblasts and stimulates production of stroma-derived factors

Endothelin (ET)-1 is a potent inducer of peptic ulcers. The roles of ET-1 in ulcer healing, however, have remained unclear, and these were investigated in mice. Gastric ulcers were induced in mice by serosal application of acetic acid. Three days later, mice were given a neutralizing ET-1 antibody or nonimmunized serum. The ulcer size, amount of fibrosis and myofibroblasts, and localization of ET-1 and ET(A/B) receptors were analyzed. To elucidate the mechanisms underlying the effects of ET-1, we examined the proliferation, migration, and release of growth and angiogenic factors in gastric myofibroblasts with or without ET-1. The expression of prepro-ET-1 (an ET-1 precursor) and ET-converting enzyme-1 was examined in gastric myofibroblasts using RT-PCR. Immunoneutralization of ET-1 delayed gastric ulcer healing. The areas of fibrosis and myofibroblasts were smaller in the anti-ET-1 antibody group than in the control. ET-1 was expressed in the gastric epithelium, myofibroblasts, and other cell types. ET(A) receptors, but not ET(B) receptors, were present in myofibroblasts. ET-1 increased proliferation and migration of gastric myofibroblasts. ET-1 stimulated the release of hepatocyte growth factor, VEGF, PGE(2), and IL-6 from gastric myofibroblasts. mRNA for prepro-ET-1 and ET-converting enzyme-1 was also expressed. ET-1 promotes the accumulation of gastric myofibroblasts and collagen fibrils at gastric ulcers. ET-1 also stimulates migration and proliferation of gastric myofibroblasts and enhances the release of growth factors, angiogenic factors, and PGE(2). Thus ET-1 has important roles not only in ulcer formation but also in ulcer healing via mobilizing myofibroblasts and inducing production of stroma-derived factors.     

The healing effects of Centella extract and asiaticoside on acetic acid induced gastric ulcers in rats

In this study, the healing effects of Centella asiatica water extract (CE) and asiaticoside (AC), an active constituent of CE, on acetic acid induced gastric ulcers (kissing ulcers) in rats were examined. CE was prepared from Centella asiatica dry plant and the concentration of AC in CE was quantitatively determined with the use of high performance liquid chromatography analysis. Different concentrations of CE and AC were orally administered to rats with kissing ulcers. They were found to reduce the size of the ulcers at day 3 and 7 in a dose-dependent manner, with a concomitant attenuation of myeloperoxidase activity at the ulcer tissues. Epithelial cell proliferation and angiogenesis were on the other hand promoted. The expression of basic fibroblast growth factor, an important angiogenic factor, was also upregulated in the ulcer tissues in rats treated with CE or AC. These results further suggest the potential use of Centella asiatica and its active ingredient as anti-gastric ulcers drugs.     

Growth factors in ulcer healing: lessons from recent studies.

Growth factors such as epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and more recently vascular endothelial growth factor (VEGF) have been used extensively to heal experimental gastric, duodenal and colonic ulcers in animal models. Encouraging results have been reported in clinical trials with EGF and bFGF. Since our laboratory has been involved with the initial ulcer healing studies with bFGF, PDGF and VEGF, we summarize here the major lessons from these studies and from literature data. These conclusions relate to the role of: 1) gastrointestinal (GI) secretion; 2) epithelial versus vascular components of the healing; 3) efficacy in the upper and lower GI tract; 4) quality of ulcer healing; as well as 5) the endogenous origin; and 6) molar potency of growth factors. Namely, among these growth factors only EGF inhibits gastric acid and stimulates duodenal bicarbonate secretion, while chronic administration of bFGF slightly enhances gastric secretion and PDGF has no effect demonstrating that potent ulcer healing can be achieved without influencing acid base and mucus secretion. This might be related to the fact that these growth factors stimulate with varying potency virtually all the cellular elements needed for ulcer healing, e.g., epithelial cell proliferation and migration by EGF > bFGF > PDGF, fibroblast proliferation by bFGF > PDGF and angiogenesis by VEGF > bFGF > PDGF > EGF. Conceptually, the most interesting results were obtained recently with VEGF which is virtually specific for angiogenesis, illustrating that stimulation of vascular factors is sufficient for ulcer healing because epithelial cells apparently spontaneously proliferate and migrate over a dense granulation tissue to complete the healing process. Since these growth factors directly stimulate the cell components of ulcer healing, it is probably not surprising that they are active in both upper and lower GI tract lesions, produce good quality of ulcer healing in comparison with spontaneously healed duodenal ulcers which are hypovascular and muscle regeneration is not part of natural healing. Contrary to other antiulcer drugs, these growth factors are endogenously derived and play a role in the natural history of ulcer healing, and since these relatively large peptides (18-45 kDa) are active in ng quantities, their molar potency is 2-7 million times superior to cimetidine-like drugs. Thus growth factors are endogenously derived very potent antiulcer drugs which act independently of GI secretion, are active in upper and lower GI lesions, and since they stimulate virtually all the cells of the healing process, they produce an excellent quality of ulcer healing.