Immunocytochemically detectable metallothionein in granulation tissue surrounding mucosal ulceration

Summary Metallothioneins (MTs) are low-molecular-weight heavy metal binding proteins which are effective free oxygen radical scavengersin vitro. Free oxygen radicals have been implicated in the pathogenesis of stress-induced acute gastric mucosal ulceration and ischaemic injury in rat and man. Experimentally, MTs can have a protective role in stress-induced ulceration in rats. The possible cytoprotective role of MTs in chronic mucosal ulceration in man has not been previously studied. Evidence for locally produced MTs in human chronic gastric and small bowel ulcers has been sought by immunocytochemical staining using a monoclonal antibody (E9) to MT. At the base of ulcers MT has been localized to spindle cells (fibroblasts) in granulation tissue. Labelling of macrophages with a pan-macrophage marker KP1, and double labelling with KP1 and E9 showed two distinct populations, and MT appeared to be localized primarily in fibroblast-like cells.     

Comparative studies on starvation - and indomethacin - induced ulcerations in albino rats.

Experimental models of chronic and acute peptic ulcerations were produced in the albino rats by means of prolonged starvation and indomethacin administration. In the case of acute indomethacin-induced peptic ulceration, the effects of anticholinergic drugs on the ulcers produced were also studied. Starving the rats for a period of seven days produced gastric ulceration in all the rats used while indomethacin produced gastric ulceration within five hours in all the rats used. Severe ulceration of the degree found in human peptic ulcer disease was produced only by chronic starvation. Anticholinergic drugs ameliorated indomethacin-induced gastric ulceration, partly at least, by reducing intra-gastric acidity.     

Localization of Human Mesenchymal Stem Cells from Umbilical Cord Blood and Their Role in Repair of Diabetic Foot Ulcers in Rats

The aim of this study is to explore the localization of human mesenchymal stem cells from umbilical cord matrix (hMSCs-UC) and the role of these cells in the repair of foot ulcerate tissue in diabetic foot ulcers in rats. A diabetic rat model was established by administering Streptozotocin. Diabetic foot ulceration was defined as non-healing or delayed-healing of empyrosis on the dorsal hind foot after 14 weeks. hMSCs-UC were delivered through the left femoral artery. We evaluated the localization of hMSCs-UC and their role in tissue repair in diabetic foot ulcers by histological analysis, PCR, and immunohistochemical staining. A model for diabetes was established in 54 out of 60 rats (90% success rate) and 27 of these rats were treated with hMSCs-UC. The area of ulceration was significantly and progressively reduced at 7 and 14 days following treatment with hMSCs-UC. This gross observation was strongly supported by the histological changes, including newly developed blood vessels and proliferation of inflammatory cells at 3 days post-treatment, significant increase in granulation tissue at 7 days post-treatment and squamous epithelium or stratified squamous epithelium at 14 days post-treatment. Importantly, human leukocyte antigen type-I (HLA-1) was confirmed in ulcerated tissue by RT-PCR. The expression of cytokeratin 19 was significantly increased in diabetic model rats, with no detectable change in cytokeratin 10. Additionally, both collagens I and III increased in model rats treated with hMSCs-UC, but the ratio of collagen I/III was less significant in treated rats compared with control rats. These results suggest that hMSCs-UC specifically localize to the target ulcerated tissue and may promote the epithelialization of ulcerated tissue by stimulating the release of cytokeratin 19 from keratinocytes and extracellular matrix formation.     

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.     

Sustained-release adrenomedullin ointment accelerates wound healing of pressure ulcers

Pressure ulcers are one of the most common complications in elderly, incontinent or paralyzed patients. For the healing of pressure ulcers, the development of granulation tissue and reepithelialization is required. Adrenomedullin (AM), an endogenous vasodilator peptide, is reported to stimulate the proliferation and migration of various cells including endothelial cells, fibroblasts and keratinocytes. Therefore, we hypothesized that AM might accelerate the healing process of pressure ulcers in which these cells were involved. We developed a sustained-release ointment containing human recombinant AM, and applied it in a mouse model of pressure ulcer twice a day for 14 days. Human AM was efficiently absorbed in wound area, but its blood concentration was negligible. AM ointment significantly reduced the wound area on day 5 to 7 after injury. In addition, AM ointment accelerated the formation of granulation tissue and angiogenesis as well as lymphangiogenesis after 7 days of treatment. Immunological analysis revealed that Ki-67-positive proliferating cells in granulation tissue expressed AM receptors. In summary, sustained-release AM significantly improved wound healing of pressure ulcers through acceleration of granulation and induction of angiogenesis and lymphangiogenesis. Therefore, sustained-release AM ointment may be a novel therapeutic agent for pressure ulcers.     

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.     

Cimetidine in patients with gastric ulcer: a multicentre controlled trial.

Forty-five adult outpatients with endoscopically confirmed gastric ulceration completed a double-blind trial of either cimetidine (1 g/day) or placebo. After six weeks 18 of the 23 patients receiving cimetidine showed complete ulcer healing compared with only six of the 22 patients receiving placebo. The cimetidine group also had fewer days with pain than the placebo group but the difference was not statistically significant. Cimetidine therefore seems to promote healing of gastric ulcers without severe side effects, although its effect on pain is less pronounced than in patients with duodenal ulcers.     

Monocyte chemotactic protein-1 regulates leukocyte recruitment during gastric ulcer recurrence induced by tumor necrosis factor-alpha

TNF-alpha has numerous biological activities, including the induction of chemokine expression, and is involved in many gastric injuries. C-C chemokines [monocyte chemotactic protein (MCP)-1 and macrophage inflammatory protein (MIP)-1alpha] and C-X-C chemokines [MIP-2 and cytokine-induced neutrophil chemoattractant (CINC)-2alpha] mediate chemotaxis of monocytes and neutrophils, respectively. We examined the roles of TNF-alpha and dynamics of chemokine expression in gastric ulceration including ulcer recurrence and indomethacin-induced injury. Rats with healed chronic gastric ulcers received intraperitoneal TNF-alpha to induce ulcer recurrence. Some rats were given neutralizing antibodies against neutrophils or MCP-1 together with TNF-alpha. In a separate experiment, rats were orally administered 20 mg/kg indomethacin with or without pretreatment with pentoxifylline (an inhibitor of TNF-alpha synthesis) or anti-MCP-1 antibody. TNF-alpha (1 microg/kg) induced gastric ulcer recurrence after 48 h, which was completely prevented by anti-neutrophil antibody. TNF-alpha increased the number of macrophages and MCP-1 mRNA expression in scarred mucosa from 4 h, whereas it increased MPO activities (marker of neutrophil infiltration) and mRNA expression of MIP-2 and CINC-2alpha from 24 h. Anti-MCP-1 antibody inhibited leukocyte infiltration with reduction of the levels of C-X-C chemokines and prevented ulcer recurrence. Indomethacin treatment increased TNF-alpha/chemokine mRNA expression from 30 min and induced macroscopic erosions after 4 h. Pentoxifylline inhibited the indomethacin-induced gastric injury with reduction of neutrophil infiltration and expression of chemokine (MCP-1, MIP-2, and CINC-2alpha). Anti-MCP-1 antibody also inhibited the injury and these inflammatory responses but did not affect TNF-alpha mRNA expression. In conclusion, increased MCP-1 triggered by TNF-alpha may play a key role in gastric ulceration by regulating leukocyte recruitment and chemokine expression.     

Prophylactic and healing properties of a new anti-ulcer compound from Enantia chlorantha in rats.

Decoctions prepared from Enantia chlorantha are used in the traditional treatment of some forms of ulcers. We have tested the anti-ulcer actions of a novel protoberberine-type alkaloid (7,8,-dihydro-8-hydroxypalmatine (1)) obtained from the bark of E. chlorantha using the HCl/ethanol, absolute ethanol and pylorus ligation techniques. The healing effect on chronic acetic acid-induced gastric ulcer was also tested. 1 (50 and 100 mg/kg) dose-dependently inhibited the formation of gastric ulcers induced by HCl/ethanol (35-52% inhibition), absolute ethanol (46-53% inhibition) and pylorus ligation (38-62% inhibition). The prophylactic actions were associated with significant increases in gastric mucus production compared with the controls. The significant inhibition of pylorus-ligated ulcers occurred at gastric acid concentrations (83 mEq/l) previously known to induce severe gastric ulceration in rats. The ulcer-healing test showed significant macromorphological and histological acceleration of healing following 10-day treatment with 1 (40 - 80 mg/kg). The results show that 1 has no anti-secretory effects. The prophylactic anti-ulcer effects are associated with enhanced mucus production, which is an important factor in the mechanism of the local healing process of chronic gastric ulcers.     

IL-21 is highly produced in Helicobacter pylori-infected gastric mucosa and promotes gelatinases synthesis

Helicobacter pylori (Hp) infection is associated with gastric inflammation and ulceration. The pathways of tissue damage in Hp-infected subjects are complex, but evidence indicates that T cell-derived cytokines enhance the synthesis of matrix metalloproteinases (MMP) that contribute to mucosal ulceration and epithelial damage. In this study, we have examined the role of the T cell cytokine IL-21 in Hp-infected gastric mucosa and evaluated whether IL-21 regulates MMP production by gastric epithelial cells. We show that IL-21 is constitutively expressed in gastric mucosa and is more abundant in biopsy specimens and purified mucosal CD3(+) T cells from Hp-infected patients compared with normal patients and disease controls. We also demonstrate that IL-21R is expressed by primary gastric epithelial cells, as well as by the gastric epithelial cell lines AGS and MKN28. Consistently, AGS cells respond to IL-21 by increasing production of MMP-2 and MMP-9, but not MMP-1, MMP-3, MMP-7, or tissue inhibitors of MMP. Analysis of signaling pathways leading to MMP production reveals that IL-21 enhances NF-kappaB but not MAPK activation, and inhibition of NF-kappaB activation reduces IL-21-induced MMP-2 and MMP-9 production. Finally, we show that treatment of Hp-infected gastric explants with anti-IL-21 reduces epithelial cell-derived MMP-2 and MMP-9 production. These data indicate that IL-21 is overexpressed in Hp-infected gastric mucosa where it could contribute to increased epithelial gelatinase production.     

Studies of the degraded carrageenan-induced colitis of rabbits. I. Changes in the epithelial glycoprotein O-acylated sialic acids associated with ulceration

Summary Ulceration of the large intestine, induced in New Zealand white rabbits by the administration of 1% w/v degraded carrageenan for periods of up to nine weeks, was studied by a combination of histochemical and chemical procedures for the identification ofO-acylated sialic acid. The onset of the disease was rapid, visible faecal blood being observed within five days. Caecal ulceration was diffuse throughout the mucosa being more prominent and frequent on the transverse folds. In some cases large, discrete ulcers were observed in the rectal portion of the lower colon, while in a small number of cases there were ulcers at the caecal end of the upper colon. The ulceration was characterized by oedema, congestion, haemorrhage, inflammation and mucosal ulceration. The inflammatory exudate consisted of macrophages, lymphocytes, a few plasma cells and, in animals treated for a long peroid, aggregates of eosinophils. The ulceration was associated with a marked reduction in the proportion of the caecal epithelial glycoprotein sialic acids which were neuraminidase-resistant and/or substituted in the side chain. Histochemical studies indicated that changes in side chain substitution were more marked at the margins of the ulcers and could be observed in sections of colon that showed no evidence of ulceration.     

Dapsone for the treatment of doxorubicin extravasation injury in the rat

Doxorubicin is the most common antitumor drug implicated in serious extravasation injuries. Progressive tissue necrosis may lead to intense pain, chronic ulceration, and disfiguring tissue loss. This progressive necrosis is analogous to that seen with brown recluse spider bites, where dapsone is an established mode of therapy, minimizing the area of tissue loss by a proposed antiinflammatory mechanism. The backs of 50 Lewis rats were injected intradermally with 1 mg of doxorubicin in 1 cc of saline to simulate an extravasation injury. The rats were divided into five groups for treatment with oral dapsone 50 mg/kg/day: 10 were controls (no treatment), 10 were started the day before injury, 10 were started the day of injury, 10 were started the day after injury, and 10 were started 1 week after injury. The area of ulceration was calculated by planimetry. The data suggest that dapsone has little positive effect on healing extravasation ulcers.     

Frozen cultured sheets of human epidermal keratinocytes enhance healing of full-thickness wounds in mice

Sheets of cultured allogeneic human keratinocytes have been used for the treatment of burns and chronic leg ulcers but there has been no animal assay for the therapeutic action of these cultures. In order to analyze the effects of frozen cultures of human keratinocytes on wound healing, we have developed such an assay based on the rate of repair of full-thickness skin wounds in immunocompetent NMR1 mice. Reepithelialization of the control wounds, originating from the murine epithelium at the edge of the wound, occurred at a constant rate of advance of 150 microm/day. When frozen cultured human epidermal sheets were thawed at room temperature for 5-10 min and applied to the surface of the wound, the murine epithelium advanced at 267 microm/day. Most wounds treated with frozen cultures completely healed after 10 days, whereas most control wounds required 16 days. The accelerated reepithelialization did not depend on the presence of proliferative human keratinocytes in the frozen cultures. The cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. This simple assay should permit quantitative analysis of the effects on healing exerted not only by cultured cells, but also by proteins and small molecules.     

Chronic gastric ulceration causes matrix metalloproteinases-9 and -3 augmentation: Alleviation by melatonin

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes capable of degradation of extracellular matrix (ECM) and key player in various inflammatory diseases. We investigated the regulation of MMPs in chronic gastric ulceration in mice. We generated chronic gastric ulcers in mice by indomethacin and examined the activity and expression of MMP-9 and -3 in stomach. Melatonin (N-acetyl-5-methoxytryptamine) treatment has also been applied to mice to characterize the changes in expression and activities of MMPs in gastric tissues. We observed significant upregulation of MMP-9 and -3 expressions and activities in stomach with increasing doses and duration of indomethacin that corroborated with increased activity of activator protein (AP)-1. Substantial damage in gastric epithelial layer was found during chronic ulceration. Melatonin suppressed MMP-9 and -3 expressions and activities during prevention and healing of chronic gastric ulcers. It also suppressed protein oxidation, lipid peroxidation and antioxidant enzymes. Additionally, expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-8 was significantly high in ulcerated stomachs while melatonin treatment blocked them to control level. We found elevated phosphorylation of extracellular-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK) during chronic gastric ulceration, which were significantly reversed by melatonin. Moreover, expression of NF-κB, c-fos and c-jun were inhibited by melatonin resulting down regulation of MMP-9 and -3 expressions. In summary, oxidative stress is preceded by chronic inflammation that enhances the expression of MMP-9 and -3, while melatonin arrests both of them via reduction of AP-1 activity during protection of ulcer.     

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.     

The protective effect of zinc sulphate pretreatment against duodenal ulcers in the rat

Exogenously administered zinc compounds have been shown to possess antiulcer activity in the development of gastric lesions. The aim of this study was to investigate the effects of zinc sulphate pretreatment of rats on cysteamine-induced duodenal ulcers and to correlate them with changes in zinc serum and tissue levels. Atomic absorption spectrophotometry was used to determine zinc serum and tissue concentrations in all animal groups. Cysteamine produced marked duodenal ulceration in control animals 24 h after application, with an increase in endogenous zinc tissue concentrations and a marked decrease in serum concentrations. Zinc sulphate (20, 40 or 80 mg kg-1) applied per os one hour prior to cysteamine application inhibited the development of duodenal lesions in a dose-related manner. The application of zinc sulphate in a single intraperitoneal (i.p.) application (80 mg kg-1) did not, however, prevent the formation of duodenal lesions. In order to assess zinc absorption from the gastrointestinal tract, one group of rats received a single oral dose of zinc sulphate (80 mg kg) without cysteamine application. The observations of this study seem to indicate that zinc plays an important cytoprotective role in duodenal ulcer disease.     

Chronic ulcer of the leg: clinical history.

Six hundred patients with chronic leg ulcers were studied by detailed history and examination as part of a population survey. In 22% ulceration began before the age of 40, and in this group the sex incidence was equal. Over age 40 there was an increasing preponderance of women. Ulcers were significantly more common in the left leg in women but not in men. The site of 26% of ulcers did not include the classical medial goiter area. The median duration of the ulceration at the time of the survey was nine months and 20% had not healed in over two years. The great majority of patients had had recurrence, 66% having had episodes of ulceration for more than five years. Healing of ulcers is a serious problem, but preventing recurrence is the greater challenge.     

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.     

A deficit in collagenase activity contributes to impaired migration of aged microvascular endothelial cells

Angiogenesis is impaired in aging. Delayed neovascularization is due, in part, to slowed endothelial cell migration. Migration requires an optimal level of adhesion to matrix proteins, a process mediated by matrix-degrading metalloproteases (MMPs) such as MMP1. To determine whether impaired angiogenesis in aging is associated with altered synthesis and activity of MMP1, we examined the expression of collagenase and tissue inhibitor of metalloprotease 1 (TIMP1) by immunostain of angiogenic sponge implants from young and aged mice. To characterize the relevance of MMP activity during the movement of aged endothelial cells, the secretion of MMP1 and TIMP1 by late-passage human microvascular endothelial cells (hmEC aged in vitro) and their non-aged (young) counterparts was quantified. The migration of aged human microvascular endothelial cells and the effect of inhibition of TIMP1 on the migration of aged hmEC or collagen I was also measured. Relative to young mice, granulation tissue from aged mice showed less expression of collagenase and increased expression of TIMP1. In vitro, aged hmEC were deficient in MMP1 secretion (55 +/- 13% relative to young cells) and activity but showed increased expression of TIMP1 (280 +/- 109% relative to young cells). Aged hmEC migrated significantly less distance than did young hmEC over a 5-day period (59 +/- 8% relative to young cells). In the presence of a blocking antibody to TIMP1, aged hmEC showed a significant increase in the distance migrated on collagen I over a 5 day period (142 +/- 11% relative to untreated aged hmEC). We propose that deficient MMP1 activity contributes to impaired cellular movement in aged microvascular endothelial cells and that perturbations that enhance collagenase activity increase their migratory ability and angiogenic potential.