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Objectives: Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that directly upregulates VEGF, Ref-1, p21, and anti-apoptotic genes such as Bcl-xL. In this study, we hypothesized that STAT3 signaling is activated and provides a critical protective role that is required for enterocyte survival during the early phases of cysteamine-induced duodenal ulcers., Methods: We studied the effect of inhibition of STAT3 activity on cysteamine-induced duodenal ulcers in rats and egr-1 knockout mice using STAT3/DNA binding assay, immunohistochemistry, immunoblot, and quantitative reverse transcriptase PCR analyses., Results: We found that G-quartet oligodeoxynucleotides T40214, a specific inhibitor of STAT3/DNA binding, aggravated cysteamine-induced duodenal ulcers in rats 2.8-fold (p < 0.05). In the pre-ulcerogenic stage, cysteamine induced STAT3 tyrosine phosphorylation, its translocation to nuclei, an increased expression and nuclear translocation of importin α and β in the rat duodenal mucosa. Cysteamine enhanced the binding of STAT3 to its DNA consensus sequences at 6, 12, and 24 h after cysteamine by 1.5-, 1.8-, and 3.5-fold, respectively, and activated the expression of STAT3 target genes such as VEGF, Bcl-xL, Ref-1, and STAT3-induced feedback inhibitor, a suppressor of cytokine signaling 3. We also demonstrated that egr-1 knockout mice, which are more susceptible to cysteamine-induced duodenal ulcers, had lower levels of STAT3 expression, its phosphorylation, expression of importin α or β, and STAT3/DNA binding than wild-type mice in response to cysteamine., Conclusions: Thus, STAT3 represents an important new molecular mechanism in experimental duodenal ulceration.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Our recent studies demonstrated a critical role of enhanced transcriptional activity of early growth response factor-1 (Egr-1) in early stages of cysteamine-induced duodenal ulcer in rats. Since cysteamine is also a reducing agent, the aims of this study were to determine the effect of cysteamine on proximal duodenal mucosa: (a) redox status, (b) mucosal oxygenation, (c) expression of hypoxia-inducible factor 1 (HIF-1alpha) and its binding to DNA, and (d) HIF-1alpha interaction with Egr-1 and other redox-sensitive transcription factors. Here we demonstrate for the first time that cysteamine treatment reduced the duodenal oxygenation by 19% (vs. baseline) and markedly increased the redox status in duodenal mucosa (p<0.05). Cysteamine increased HIF-1alpha expression, its binding to DNA, and enhanced the HIF-1alpha interactions with Egr-1 and other transcription factors (e.g., AP-1, AP-2, L-III BP, NF-E1, NF-E2, STAT4, and MRE), their binding to DNA. Thus, these data demonstrate the involvement of the redox-dependent regulatory mechanisms in the early stages of duodenal ulceration.

Recent studies from our laboratory demonstrated that Egr-1 is upregulated in the rat duodenal mucosa during cysteamine-induced duodenal ulceration and that antisense egr-1 oligonucleotide aggravates the duodenal ulcers. This study was aimed to determine the effects of cysteamine on redox-sensitive Egr-1 transcriptional activity and on other thiol-containing proteins such as redox factor-1 (Ref-1) and thioredoxin (Trx). Here we demonstrate for the first time that cysteamine increases the expression and nuclear translocation of Egr-1, Ref-1, and Trx, and activates binding of Egr-1 to DNA. Moreover, we also show that Egr-1 forms a complex with other redox-sensitive transcription factors (e.g., AP-1, AP-2, NFATc, Sp1, PAX-5, MTF-1, c-Myb, and CREB) in rat duodenal mucosa and that cysteamine enhances the formation of these complexes. The antioxidant ebselen markedly elevated the nuclear Ref-1 expression and Egr-1/DNA binding, and decreased the ulcerogenic effect of cysteamine as did catalase. Thus, redox-sensitive signaling systems seem to play an important role in cysteamine-induced duodenal ulceration.

The complete purification of the first angiogenic molecule, basic fibroblast growth factor (bFGF), was carried out in the authors' laboratory in 1983. Application of this peptide to chronic wounds enhances angiogenesis and accelerates wound healing. The authors showed that an acid-stable form of bFGF (i.e., bFGF-CS23) could be administered orally to rats with duodenal ulcers. The peptide promoted a ninefold increase of angiogenesis in the ulcer bed and accelerated ulcer healing more potently than cimetidine. Basic fibroblast growth factor did not reduce gastric acid. The authors now show that bFGF exists as a naturally occurring peptide in rat and human gastric and duodenal mucosa. This endogenous bFGF is present also in the bed of chronic ulcers in rats. Sucralfate binds bFGF and protects it from acid degradation. The sucralfate is angiogenic, based on its affinity for bFGF. When sucralfate is administered orally to rats, it significantly elevates the level of bFGF in the ulcer bed. Cimetidine, by its capacity to reduce gastric acid, also elevates bFGF in the ulcer bed. A hypothetical model is proposed in which prevention of ulcer formation or accelerated healing of ulcers by conventional therapies may be FGF dependent. Acid-stable bFGF-CS23 may be considered as a form of replacement therapy in the treatment of duodenal ulcers.

This review provides evidence that gastroduodenal mucosal injury is a complex process because of the heterogenous structure and multiple functions of the gut. The action of exogenous etiologic agents is usually mediated in part or amplified by endogenous mediators that very often exert biphasic, i.e., damaging and protective, effects. The pathogenetic pathways involved are direct/indirect chemical injury, vascular damage and its consequences, and acute or chronic inflammatory processes following infectious, chemical, or ischemic injury. The role of oxygen, free radicals, calcium, and proteases as well as the components and forms of gastroduodenal injury, e.g., reversible and irreversible cell injury, tissue necrosis, and acute and chronic inflammation, are also briefly discussed.

Acute duodenal ulcer produced by subcutaneous injection of propionitrile in rats was studied by stereo, light, and electron microscopy in order to gain insight into the localization and mechanism of initial cell injury. Stereomicroscopy revealed an initial fissuring and splitting of the tips of the villus folds within 4 hours after two injections of propionitrile. This was followed by sloughing of the epithelium, shortening and effacement of the villus folds, and within 24 hours the appearance of discrete ulcers in the mucosa of the proximal duodenum. In most of the rats, two ulcers developed: the first and larger ulcer was on the antimesenteric side of the duodenum, and the other, a small and more superficial one, was on the opposite wall. Ultrastructural lesions appeared in the absorptive epithelial cells of the proximal duodenum within 5 hours following a single dose of propionitrile. The cytoplasmic changes of cellular injury were preceded and/or accompanied by beading and loss of microvilli. The initiation of propionitrile-induced alterations at the tip of the villi in the proximal duodenum suggests that the ulcerogenic agent acts from the luminal side of the duodenum and probably originates from the stomach.

Cysteamine and propionitrile cause severe duodenal ulcers with perforation within 24-48 h after a single injection in rats. These animal models were used to gain insight into the early, preulcerogenic biochemical changes in the duodenal mucosa. The results indicate that a single sc injection of cysteamine and propionitrile induced dose- and time-dependent decreases in the activity of phosphoprotein phosphatase (PPPase) in homogenate and particulate fractions of rat duodenal mucosa. The decrease in enzyme activity was detectable 4 h after the injection of the ulcerogens, it was maximal at 12 h, and hardly detectable at 24 h. No effect on the enzyme activity was found under in vitro conditions. PPPase activity in the liver was not influenced by either cysteamine or propionitrile. Furthermore, the toxic but nonulcerogenic derivative of cysteamine ethanolamine had no effect on PPPase in the duodenum. Thus, the effect of the duodenal ulcerogens on PPPase activity was indirect and organ specific, related only to the target organ (i.e., duodenal mucosa). The effect of the drugs was also selective at the level of mucosal cells: both duodenal ulcerogens depleted protein and alkaline phosphatase but not lysosomal acid phosphatase. The decrease of PPPase activity could be a general property of the duodenal ulcerogens since it is independent of their effect on endogenous somatostatin.

Neutralization of acid was evaluated in rat proximal duodenal segments isolated from biliary and pancreatic secretions. Duodenal ulcerogenic doses of cysteamine produced a significant decrease in acid disposal 0.5-2 hr after treatment. Oral or subcutaneous administration of the duodenal ulcerogen was effective. The potent ulcerogen cysteamine produced a more pronounced decrease than propionitrile (a weak duodenal ulcerogen). The failure of ethanolamine, a nonulcerogenic structural analog of cysteamine to significantly alter acid disposal suggests that the effect is not due to the toxic properties of the duodenal ulcerogen. The results reinforce the concept that the duodenum is able to dispose of significant quantities of acid. The decrease in acid-handling may contribute to duodenal susceptibility to acid after treatment with ulcerogens and possibly reflects pathophysiologic changes early in duodenal ulceration.

Previous structure-activity and pharmacologic studies with duodenal ulcerogens cysteamine and propionitrile implicating catecholamines in the pathogenesis of duodenal ulceration have now been followed up by dose- and time-response biochemical investigations to assess the importance of monoamines in the development of duodenal ulcers. The concentrations of norepinephrine (noradrenaline), dopamine, serotonin and their metabolites were measured in total brain, brain regions, stomach, duodenum, pancreas and adrenals in the rat. Turnover of catecholamines was determined in rats pretreated with the inhibitor of tyrosine hydroxylase alpha-methyl-p-tyrosine. The duodenal ulcerogens caused a dose- and time-dependent depletion of norepinephrine in virtually all the tissues examined. The effect was maximal 4 or 7 hr after cysteamine or propionitrile, and norepinephrine levels returned to normal in 24 hr. Dopamine changes were selective and often biphasic, e.g., elevation in adrenals, biphasic in brain cortex, hippocampus and midbrain, but uniformly decreasing in glandular stomach and duodenum. In the median eminence dopamine levels decreased by 181 and 324% at 15 and 30 min, respectively, after cysteamine, but neither dopamine nor 3,4-dihydroxyphenylacetic acid was modified in the periventricular nucleus. Serotonin levels were relatively stable, revealing slight elevations or no changes in most of the tissues. The turnover of norepinephrine was accelerated by both chemicals in virtually all brain regions, but dopamine turnover was affected only in a few areas, e.g., in the corpus striatum and medulla oblongata cysteamine decreased dopamine turnover, whereas propionitrile first (at 1 hr) accelerated then (at 8 hr) significantly suppressed it.(ABSTRACT TRUNCATED AT 250 WORDS)

Duodenal ulcers were induced acutely in female rats by a single oral administration of cysteamine, 70 mg/100 gm, in order to study morphologic progression of lesion development from the perspective of cellular surface changes by scanning electron microscopy. Thick sections of resin-embedded specimens were also studied by light microscopy, and animals were sacrificed at intervals of 30 minutes, 1, 2, 4, 8, 12, 20, and 24 hours post-treatment. Earliest evidence of cytologic lesions was apparent at 2 hours and data confirmed earlier reports that alterations began at villous tips. Both cellular sloughing and in situ cellular injury were evident, the latter phenomenon constituting the principal mode of cysteamine-induced erosion. In situ change began, from surface perspective, as a minute cavitation on the apical aspect of an isolated, single epithelial cell which was surrounded by normal cells. These early lesions progressed to in situ necrosis either of isolated cells or of small clusters of adjacent cells. This phenomenon occurred concurrently on multiple villi, all within the localized site at which cysteamine-induced duodenal ulcers are known to develop. An additional early morphologic change was the occasional appearance of a background of pleomorphic cellular apices of variable size on the villous ridges. By 8 to 12 hours, cellular damage advanced to erosions with some cells in the preulcer area still showing initial stages of in situ cellular injury. Precipitated mucus on the surface was increased in the preulcer area, and by 20 to 24 hours typical duodenal ulcers were evident. These scanning electron microscopic data confirm the significance of surface damage at villous tips very early in the cysteamine-induced ulcerogenesis. The present higher resolution findings demonstrated that earliest cellular damage, principally in situ cell injury, occurred simultaneously at multiple sites in the preulcer zone rather than at a single cluster of cellular damage which enlarges peripherally.

Increased gastric acid secretion, enhanced acid delivery to the duodenum, and reduced alkaline secretion in the proximal duodenum are relatively well-established pathophysiologic abnormalities in duodenal ulcer. Impaired duodenal motility, however, may also contribute to duodenal ulceration by altering the distribution of acid and alkaline secretions along the upper digestive tract. We tested the hypothesis that the duodenal ulcerogens cysteamine, MPTP, and mepirizole modify duodenal motility in the rat and that motility changes might be a common and early alteration in experimental duodenal ulceration. All three duodenal ulcerogens rapidly produced extensive changes in duodenal myoelectric activity and reduced the frequency of myoelectric slow waves. Cysteamine induced marked hypermotility for at least 6 hr; MPTP rapidly decreased motility and fragmented the myoelectric migrating pattern. Mepirizole induced biphasic changes: an early hypermotility phase of about 30 min was followed by profound hypomotility. These results indicate that marked alterations of duodenal motility are common during experimental duodenal ulceration. In light of the differential effect of the ulcerogens on duodenal motility, it remains to be determined how these changes influence acid neutralization in the proximal duodenum. Nevertheless, our results suggest that all three duodenal ulcerogens, which are different in structure, alter duodenal motility.

A single dose of acrylonitrile can produce fatal adrenal apoplexy within approximately 2 h. Our previous studies also indicate that multiple injections of the chemical cause acute hemorrhagic and occasional nonperforating duodenal ulcers. Other authors have reported increase in gut and lung neoplasia after chronic exposure. The present study was designed to elucidate the subacute and chronic actions of acrylonitrile on the adrenals, stomach and duodenum by correlating biochemical, functional and morphologic investigations, as well as to gain insight into the mechanisms of action of acrylonitrile. Rats were exposed to 0, 0.0001% (1 ppm), 0.002%, 0.01%, 0.05% or 0.2% acrylonitrile in drinking water, or to the same amount of the chemical given through daily gavage, for 7, 21 or 60 days. Acrylonitrile caused a time- and dose-dependent decrease in plasma corticosterone levels; aldosterone was affected only by the 'high' dose and prolonged time of exposure. Young rats were more susceptible than adults to this action of acrylonitrile. The adrenal cortex, especially the zona fasciculata, was atrophic in rats that had ingested the nitrile through drinking water. At 0.05% and 0.2%, it also caused decreased food intake and body weight gain. The adrenals were enlarged with a hyperplastic zona fasciculata after daily doses of a bolus of acrylonitrile. Ingestion of the chemical did not interfere with compensatory enlargement of the adrenal gland following unilateral adrenalectomy. On the other hand, the ACTH-induced elevation of corticosterone plasma concentration was significantly attenuated by acrylonitrile in drinking water. Electron microscopy of the adrenal glands revealed no consistent changes in the steroid-producing cells. We thus postulate that accelerated turnover of circulating corticoids and/or interference with the secretion or action of ACTH may primarily be responsible for the decreased plasma levels of corticosterone and aldosterone in rats that ingest acrylonitrile. The mucosa in the stomach at the junction of the forestomach and glandular region of animals that had ingested acrylonitrile was hyperplastic. The corpus also showed regional mucosal hyperplasia with the appearance of 'cobble-stoning'. These changes were preceded and associated with an elevated concentration of non-protein sulfhydryls mostly in the mucosa of the glandular stomach. A similar, less prominent elevation also occurred in the proximal duodenum. These alterations may resemble the preneoplastic combination of elevated glutathione and focal hyperplasia described in the liver with hepatocarcinogens.(ABSTRACT TRUNCATED AT 400 WORDS)

This review is based on results from our laboratory and those published by others, and is focused on the early stages of pathogenesis that can be studied mostly in animals. Gastric mucosal injury is analysed on the examples of ethanol- and aspirin-induced lesions. Ethanol (50-100%) rapidly penetrates the mucosa, causes directly and/or indirectly (e.g., release of vasoactive products) endothelial damage in superficial and deep capillaries and venules. The vascular damage results in increased vascular permeability and decrease in blood flow leading to complete circulatory standstill in superficial capillaries 1-2 min after intragastric administration of concentrated ethanol. The direct chemical damage to surface mucosal epithelium is then followed by hypoxia and deep hemorrhagic necrosis in 1-5 min (erosion or ulcer). Unionized aspirin initiates a similar and complex yet slower progressing and less extensive erosion than alcohol. Duodenal erosion and ulcer produced by cysteamine, mepirizole or MPTP are preceded by excess acid in the proximal duodenum. This could be due to increased gastric acid output (1-4 hr), decreased bicarbonate secretion or duodenal dysmotility (0.5-8 hr) preventing the proper mix of acid and base in duodenal bulb. Necrosis and desquamation of absorptive cells in duodenal villi are evident 2-4 hr, followed by villus amputation (4-8 hr), erosion and ulcer (8-24 hr). The pathogenesis of gastroduodenal mucosal injury can thus be reconstructed from results obtained with animal models and from human studies. The results should serve as a basis to design protective drugs that are active on the basis of pathogenetic events.

Dopamine and norepinephrine concentrations in the stomach, duodenum, and brain were measured after subcutaneous injection of tyrosine methyl ester in the rat. Tyrosine significantly increased dopamine concentrations in each tissue without altering norepinephrine levels. The increase in dopamine concentration in the stomach or duodenum was observed 1 h after tyrosine methyl ester injection, but it returned to normal 4 h after the administration of the chemical.

The early morphologic sequelae induced by the duodenal ulcerogen, cysteamine, have been studied in rats by transmission electron microscopy. Cysteamine was administered per os at 70 mg/100 g body wt to groups of female rats sacrificed at 30 min, 1, 2, 4, 8, 12, 20, and 24 hr after chemical treatment, and duodenal tissue sampled from the antimesenteric side of the proximal duodenum, where ulcers develop, was studied. Emphasis was placed on early times as our previous scanning electron microscopic data had demonstrated enhanced in situ cellular necrosis and surface cavitation at 2-4 hr after cysteamine treatment. Results indicated intracellular changes as early as 30 min after treatment and prior to damage of the columnar cell microvilli or epithelial tight junctions. A staging of observed cellular degenerative changes suggested early apical endoplasmic reticular swelling and loss of cytoplasmic ground substance, followed later by moderate internal disruption of mitochondria. Through these stages the cell surface microvilli remained morphologically normal. Subsequently, microvilli degenerated and mitochondrial fine structure became severely disrupted and cell contents were expelled. Deeper villous changes such as separation of columnar cells from the lamina propria and alterations of selected elements within the lamina propria were observed. These data suggest that intracellular cytotoxic reactions at the villous tips occur early and may precede the influence of intraluminal damaging factors induced by cysteamine.

Many secretory studies reported an increase in gastric acid secretion by the duodenal ulcerogen cysteamine. A detailed analysis of these experiments, especially the results from rats with chronic gastric fistula suggest that direct stimulation of gastric acid secretion may not be the primary mechanism of the duodenal ulcerogenic action of cysteamine. We used a different approach and measured the pH at the site of ulceration in the proximal duodenum. A duodenal ulcerogenic dose of cysteamine did not change the pH at the anterior or posterior wall of the duodenum during 4 hr. In the same dose and by the same route of administration, cysteamine nevertheless induced duodenal ulcers in 24 hr. These experiments demonstrate that in addition to the effect on gastric acid secretion, other factors are needed to the effect on gastric acid secretion, other factors are needed to explain the early duodenal ulcerogenic action of cysteamine.

The effects of the duodenal ulcerogens cysteamine and propionitrile on gastroduodenal myoelectric activity and intraluminal pressure in the fasted rat as well as on contractility of isolated gut muscle strips were investigated. Duodenal ulcerogens, unlike the nonulcerogen but toxic analogue ethanolamine, caused an early disruption of the myoelectric migrating complex, a marked increase in the spiking activity, and a decrease in the frequency of the slow waves in the duodenum. Both the increased spiking activity and the decreased slow wave frequency were dose dependent for cysteamine. Manometrically recorded contractions at the stomach corpus, midantrum, and antropyloric region as well as in the proximal duodenum of the conscious rat showed decreased contractions at the corpus and midantrum and an increase at the pyloric and duodenal sites during an intravenous infusion of cysteamine. In vitro studies demonstrated that circularly or longitudinally cut muscle strips taken from different regions of the stomach and duodenum responded to cysteamine with increased contractility. In summary, the duodenal ulcerogens cysteamine and propionitrile rapidly induce motor abnormalities in the stomach and duodenum of the rat. In vitro studies suggest that a cholinergic mechanism may be involved. It is possible that motor changes play a role in the pathogenesis of the experimentally induced duodenal ulcers.

The role of endogenous somatostatin in the pathogenesis of duodenal was investigated in the present study by using the cysteamine animal model of the disease. Our previous studies showed a rapid and multiorgan depletion of somatostatin immunoreactivity (SIR) in rats given a single dose of duodenal ulcerogen cysteamine. We now determined whether acetylcholinergic and dopaminergic modulation (both known to influence the development of duodenal ulcer) are accompanied by modification of cysteamine-induced SIR depletion in rat organs. Vagotomy performed either 1 or 18 h before cysteamine administration did not interfere with the chemically induced SIR decrease in pancreas, gastric and duodenal mucosa. Vagal denervation alone had no marked influence on SIR levels but if combined with cysteamine, the SIR depletion in the stomach was significantly more pronounced than after the duodenal ulcerogen alone. Pretreatment with the dopamine agonists bromocriptine or lergotrile (known to prevent the chemically induced duodenal ulcers) did not influence the SIR depletion by cysteamine. Thus cysteamine depletes endogenous somatostatin in peripheral organs (e.g., stomach, duodenum, pancreas) by mechanisms independent of both vagus nerve and dopamine agonists. A role of central somatostatin depletion leading to disinhibition of vagus is also considered in the pathogenesis of experimental duodenal ulcer.