Indian Hedgehog mediates gastrin-induced proliferation in stomach of adult mice.

BACKGROUND & AIMS: Loss of expression of Sonic Hedgehog (Shh) from parietal cells results in hypergastrinemia in mice, accompanied by increased expression of Indian Hedgehog (Ihh) and hyperproliferation of surface mucous cells. We investigated whether hypergastrinemia induces gastric epithelial proliferation by activating Ihh signaling in mice. METHODS: We studied mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and hypergastrinemia, crossed with gastrin-deficient (GKO) mice (PC-Shh(KO)/GKO). When mice were 3-4 months old, gastric tissues were collected and analyzed by histology, for incorporation of bromodeoxyuridine, and for expression of the surface mucous cell marker Ulex europaeus. PC-Shh(KO)/GKO mice were given gastrin infusions for 7 days; gastric surface epithelium was collected and expression of Ihh was quantified by laser capture microdissection followed by quantitative reverse transcriptase polymerase chain reaction. Mouse stomach-derived organoids were incubated with or without inhibitors of WNT (DKK1) or Smoothened (vismodegib) and then cocultured with immortalized stomach mesenchymal cells, to assess proliferative responses to gastrin. RESULTS: Gastric tissues from PC-Shh(KO)/GKO mice with hypergastrinemia had an expanded surface pit epithelium, indicated by a significant increase in numbers of bromodeoxyuridine- and Ulex europaeus-positive cells, but there was no evidence for hyperproliferation. Gastrin infusion of PC PC-Shh(KO)/GKO mice increased expression of Ihh and proliferation within the surface epithelium compared with mice given infusions of saline. In gastric organoids cocultured with immortalized stomach mesenchymal cells, antagonists of WNT and Smoothened inhibited gastrin-induced proliferation and WNT activity. Activity of WNT in media collected from immortalized stomach mesenchymal cells correlated with increased expression of glioma-associated oncogene homolog 1, and was inhibited by DKK1 or vismodegib. CONCLUSIONS: Ihh signaling mediates gastrin-induced proliferation of epithelial cells in stomachs of adult mice.

The role of plasminogen activator inhibitor-1 in gastric mucosal protection.

Gastric mucosal health is maintained in response to potentially damaging luminal factors. Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) disrupt protective mechanisms leading to bleeding and ulceration. The plasminogen activator system has been implicated in fibrinolysis following gastric ulceration, and an inhibitor of this system, plasminogen activator inhibitor (PAI)-1, is expressed in gastric epithelial cells. In Helicobacter pylori-negative patients with normal gastric histology taking aspirin or NSAIDs, we found elevated gastric PAI-1 mRNA abundance compared with controls; the increase in patients on aspirin was independent of whether they were also taking proton pump inhibitors. In the same patients, aspirin tended to lower urokinase plasminogen activator mRNA. Immunohistochemistry indicated PAI-1 localization to epithelial cells. In a model system using MKN45 or AGS-GR cells transfected with a PAI-1 promoter-luciferase reporter construct, we found no evidence for upregulation of PAI-1 expression by indomethacin, and, in fact, cyclooxygenase products such as PGE2 and PGI2 weakly stimulated expression. Increased gastric PAI-1 mRNA was also found in mice following gavage with ethanol or indomethacin, but plasma PAI-1 was unaffected. In PAI-1(-/-) mice, gastric hemorrhagic lesions in response to ethanol or indomethacin were increased compared with C57BL/6 mice. In contrast, in PAI-1-H/Kß mice in which PAI-1 is overexpressed in parietal cells, there were decreased lesions in response to ethanol and indomethacin. Thus, PAI-1 expression is increased in gastric epithelial cells in response to mucosal irritants such as aspirin and NSAIDs probably via an indirect mechanism, and PAI-1 acts as a local autoregulator to minimize mucosal damage.

Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori.

BACKGROUND & AIMS: Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection. METHODS: Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis. RESULTS: Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1ß in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice. CONCLUSIONS: H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.

Inhibitors of acid secretion can benefit gastric wound repair independent of luminal pH effects on the site of damage.

BACKGROUND AND AIMS: The authors' goal was to measure pH at the gastric surface (pH0) to understand how acid secretion affects the repair of microscopic injury to the gastric epithelium. METHODS: Microscopic gastric damage was induced by laser light, during confocal/two-photon imaging of pH-sensitive dyes (Cl-NERF, BCECF) that were superfused over the mucosal surface of the exposed gastric corpus of anaesthetised mice. The progression of repair was measured in parallel with pH0. Experimental conditions included varying pH of luminal superfusates, and using omeprazole (60 mg/kg ip) or famotidine (30 mg/kg ip) to inhibit acid secretion. RESULTS: Similar rates of epithelial repair and resting pH0 values (∼pH 4) were reported in the presence of luminal pH 3 or pH 5. Epithelial repair was unreliable at luminal pH 2 and pH0 was lower (2.5±0.2, P <0.05 vs pH 3). Epithelial repair was slower at luminal pH 7 and pH0 was higher (6.4±0.1, P<0.001). In all conditions, pH0 increased adjacent to damage. At luminal pH 3 or pH 7, omeprazole reduced maximal damage size and accelerated epithelial repair, although only at pH 3 did omeprazole further increase surface pH above the level caused by imposed damage. At luminal pH 7, famotidine also reduced maximal damage size and accelerated epithelial repair. Neither famotidine nor omeprazole raised plasma gastrin levels during the time course of the experiments. CONCLUSIONS: Epithelial repair in vivo is affected by luminal pH variation, but the beneficial effects of acutely blocking acid secretion extend beyond simply raising luminal and/or surface pH.

Gastrin stimulates expression of plasminogen activator inhibitor-1 in gastric epithelial cells.

Plasminogen activator inhibitor (PAI)-1 is associated with cancer progression, fibrosis and thrombosis. It is expressed in the stomach but the mechanisms controlling its expression there, and its biological role, are uncertain. We sought to define the role of gastrin in regulating PAI-1 expression and to determine the relevance for gastrin-stimulated cell migration and invasion. In gastric biopsies from subjects with elevated plasma gastrin, the abundances of PAI-1, urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNAs measured by quantitative PCR were increased compared with subjects with plasma concentrations in the reference range. In patients with hypergastrinemia due to autoimmune chronic atrophic gastritis, there was increased abundance of PAI-1, uPA, and uPAR mRNAs that was reduced by octreotide or antrectomy. Immunohistochemistry revealed localization of PAI-1 to parietal cells and enterochromaffin-like cells in micronodular neuroendocrine tumors in hypergastrinemic subjects. Transcriptional mechanisms were studied by using a PAI-1-luciferase promoter-reporter construct transfected into AGS-G(R) cells. There was time- and concentration-dependent increase of PAI-1-luciferase expression in response to gastrin that was reversed by inhibitors of the PKC and MAPK pathways. In Boyden chamber assays, recombinant PAI-1 inhibited gastrin-stimulated AGS-G(R) cell migration and invasion, and small interfering RNA treatment increased responses to gastrin. We conclude that elevated plasma gastrin concentrations are associated with increased expression of gastric PAI-1, which may act to restrain gastrin-stimulated cell migration and invasion.

A multi-faceted intervention to reduce alcohol misuse and harm amongst sports people in Ireland: A controlled trial.

INTRODUCTION AND AIMS: Alcohol misuse and harm are more prevalent amongst sports people than non-sports people. Few studies have trialled interventions to address alcohol misuse for this group. The study aimed to test the effectiveness of an intervention to reduce alcohol misuse and related harms amongst amateur sports people in Ireland. DESIGN AND METHODS: A controlled trial was conducted in two counties in Ireland. A random selection of sports clubs in one county received a 4 month multi-faceted intervention. All sports clubs in a non-adjacent county acted as control sites. Consumption of more than 21 units of alcohol per week and six or more standard drinks on a single occasion at least once per week was the primary study outcome. Alcohol Use Disorders Identification Test scores and number of alcohol-related harms were also reported. Outcomes were assessed for cross-sectional samples of players at pre-intervention and post-intervention and paired samples of players who completed surveys at both times. Generalised linear mixed model analysis was used. RESULTS: There was no evidence of effect for the primary outcomes or Alcohol Use Disorders Identification Test scores. There was a statistically significant difference in the median number of alcohol-related harms reported by intervention group players compared with control group players at post-intervention for the paired samples [intervention: 0; control: 3; incident rate ratio 0.56 (0.37, 0.84); P = 0.005]. DISCUSSION AND CONCLUSIONS: Intervention in community sports clubs may be effective in reducing the number of alcohol-related harms. Low levels of intervention participation and inadequate intervention dose are possible reasons for lack of a broader intervention effect. [O'Farrell A, Kingsland M, Kenny S, Eldin N, Wiggers J, Wolfenden L, Allwright S. A multi-faceted intervention to reduce alcohol misuse and harm amongst sports people in Ireland: A controlled trial. Drug Alcohol Rev 2018;37:14-22].

Plasminogen activator inhibitor (PAI)-1 suppresses inhibition of gastric emptying by cholecystokinin (CCK) in mice.

The intestinal hormone cholecystokinin (CCK) delays gastric emptying and inhibits food intake by actions on vagal afferent neurons. Recent studies suggest plasminogen activator inhibitor (PAI)-1 suppresses the effect of CCK on food intake. In this study we asked whether PAI-1 also modulated CCK effects on gastric emptying. Five minute gastric emptying of liquid test meals was studied in conscious wild type mice (C57BL/6) and in transgenic mice over-expressing PAI-1 in gastric parietal cells (PAI-1H/Kß mice), or null for PAI-1. The effects of exogenous PAI-1 and CCK8s on gastric emptying were studied after ip administration. Intragastric peptone delayed gastric emptying in C57BL/6 mice by a mechanism sensitive to the CCK-1 receptor antagonist lorglumide. Peptone did not delay gastric emptying in PAI-1-H/Kß mice. Exogenous CCK delayed gastric emptying of a control test meal in C57BL/6 mice and this was attenuated by administration of PAI-1; exogenous CCK had no effect on emptying in PAI-1-H/Kß mice. Prior administration of gastrin to increase gastric PAI-1 inhibited CCK-dependent effects on gastric emptying in C57BL/6 mice but not in PAI-1 null mice. Thus, both endogenous and exogenous PAI-1 inhibit the effects of CCK (whether exogenous or endogenous) on gastric emptying. The data are compatible with emerging evidence that gastric PAI-1 modulates vagal effects of CCK.

Gastric expression of plasminogen activator inhibitor (PAI)-1 is associated with hyperphagia and obesity in mice.

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H(+)/K(+)ß subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kß mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kß mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kß mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kß mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1-null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake.

Alcohol use among amateur sportsmen in Ireland.

BACKGROUND: The objective of this study was to establish baseline data on alcohol consumption patterns, behaviours and harms among amateur sportsmen in the Republic of Ireland. FINDINGS: The study presents findings from the baseline survey for a cluster randomised controlled trial to evaluate the effectiveness of a community intervention programme to reduce problem alcohol use among a representative sample of Gaelic Athletic Association (GAA) clubs in two counties in the Republic of Ireland. Self reported alcohol use, prevalence of binge drinking, AUDIT scores and alcohol-related harms were assessed in amateur GAA sportsmen aged 16 years and over.Nine hundred and sixty (960) players completed questionnaires (72% response rate). Mean age was 24.0 years (S.D. 5.2). Of those aged 18 years or over, 75% had post-primary education; most (864, 90%) were current drinkers and 8.2% were regular smokers. The self-reported average yearly alcohol consumption was 12.5 litres. Almost one third (31%) of current drinkers reported drinking over the recommended limit of 21 standard drinks per week and just over half (54.3%) reported drinking 6 or more standard drinks in a row at least once a week (regular binge drinking). Of those who (self) completed the Alcohol Use Disorder Identification Test (AUDIT) questionnaire, three-quarters (74.7%) had a score of 8 or more; 11.5% had a score of 20 or above warranting referral for diagnostic evaluation and treatment. Almost all (87.6%) of the 864 drinkers reported experiencing at least one harm due to their drinking. These alcohol misuse outcomes were higher than those found in a nationally representative sample of males of a similar age. There were strong associations between regular binge drinking and reporting harms such as being in a fight (adjusted odds ratio (OR) 2.02, p < 0.001), missing time from work or college (adjusted OR 1.39, p = 0.04) or being in an accident (adjusted OR 1.78, p = 0.04). CONCLUSIONS: These male amateur sportsmen reported high rates of alcohol consumption and alcohol-related harm.

Gastrin activates paracrine networks leading to induction of PAI-2 via MAZ and ASC-1.

The gastric hormone gastrin regulates the expression of a variety of genes involved in control of acid secretion and also in the growth and organization of the gastric mucosa. One putative target is plasminogen activator inhibitor-2 (PAI-2), which is a component of the urokinase activator system that acts extracellularly to inhibit urokinase plasminogen activator (uPA) and intracellularly to suppress apoptosis. Previous studies have demonstrated that gastrin induces PAI-2 both in gastric epithelial cells expressing the gastrin (CCK-2) receptor and, via activation of paracrine networks, in adjacent cells that do not express the receptor. We have now sought to identify the response element(s) in the PAI-2 promoter targeted by paracrine mediators initiated by gastrin. Mutational analysis identified two putative response elements in the PAI-2 promoter that were downstream of gastrin-activated paracrine signals. One was identified as a putative MAZ site, mutation of which dramatically reduced both basal and gastrin-stimulated responses of the PAI-2 promoter by a mechanism involving PGE(2) and the small GTPase RhoA. Yeast one-hybrid screening identified the other as binding the activating signal cointegrator-1 (ASC-1) complex, which was shown to be the target of IL-8 released by gastrin. RNA interference (RNAi) knockdown of two subunits of the ASC-1 complex (p50 and p65) inhibited induction of PAI-2 expression by gastrin. The data reveal previously unsuspected transcriptional mechanisms activated as a consequence of gastrin-triggered paracrine networks and emphasize the elaborate and complex cellular control mechanisms required for a key component of tissue responses to damage and infection.

Increased expression of the urokinase plasminogen activator system by Helicobacter pylori in gastric epithelial cells.

The gastric pathogen Helicobacter pylori (H. pylori) is linked to peptic ulcer and gastric cancer, but the relevant pathophysiological mechanisms are unclear. We now report that H. pylori stimulates the expression of plasminogen activator inhibitor (PAI)-1, urokinase plasminogen activator (uPA), and its receptor (uPAR) in gastric epithelial cells and the consequences for epithelial cell proliferation. Real-time PCR of biopsies from gastric corpus, but not antrum, showed significantly increased PAI-1, uPA, and uPAR in H. pylori-positive patients. Transfection of primary human gastric epithelial cells with uPA, PAI-1, or uPAR promoters in luciferase reporter constructs revealed expression of all three in H+/K+ATPase- and vesicular monoamine transporter 2-expressing cells; uPA was also expressed in pepsinogen- and uPAR-containing trefoil peptide-1-expressing cells. In each case expression was increased in response to H. pylori and for uPA, but not PAI-1 or uPAR, required the virulence factor CagE. H. pylori also stimulated soluble and cell surface-bound uPA activity, and both were further increased by PAI-1 knockdown, consistent with PAI-1 inhibition of endogenous uPA. H. pylori stimulated epithelial cell proliferation, which was inhibited by uPA immunoneutralization and uPAR knockdown; exogenous uPA also stimulated proliferation that was further increased after PAI-1 knockdown. The proliferative effects of uPA were inhibited by immunoneutralization of the EGF receptor and of heparin-binding EGF (HB-EGF) by the mutant diphtheria toxin CRM197 and an EGF receptor tyrosine kinase inhibitor. H. pylori induction of uPA therefore leads to epithelial proliferation through activation of HB-EGF and is normally inhibited by concomitant induction of PAI-1; treatments directed at inhibition of uPA may slow the progression to gastric cancer.

Increased gastric expression of MMP-7 in hypergastrinemia and significance for epithelial-mesenchymal signaling.

Chronic hypergastrinemia is associated with enterochromaffin-like (ECL) cell hyperplasia, which may progress to gastric carcinoid tumors. The latter consists of epithelial cells and stroma, and both compartments usually regress after normalization of hypergastrinemia. We previously showed that matrix metalloproteinase (MMP)-7 in gastric epithelial cells was upregulated by Helicobacter pylori and described MMP-7-dependent reciprocal signaling between the epithelium and a key stromal cell type, the myofibroblast. Here, we describe the regulation of gastric MMP-7 by gastrin and the potential significance for recruiting and maintaining myofibroblast populations. Biopsies of the gastric corpus and ECL cell carcinoid tumors were obtained from hypergastrinemic patients. Western blot analysis, ELISA, immunohistochemistry, and promoter-luciferase (luc) reporter assays were used to study MMP-7 expression. Gastric myofibroblasts were identified by alpha-smooth muscle actin (alpha-SMA) expression, and the effects of MMP-7 on myofibroblast proliferation were investigated. In hypergastrinemic patients, there was an increased abundance of MMP-7 and alpha-SMA in gastric corpus biopsies and ECL cell carcinoid tumors. In the latter, MMP-7 was localized to ECL cells but not stromal cells, which were nevertheless well represented. Gastrin stimulated MMP-7-luc expression in both AGS-G(R) and primary human gastric epithelial cells. Conditioned medium from gastrin-treated human gastric glands stimulated myofibroblast proliferation, which was inhibited by neutralizing antibodies to MMP-7. MMP-7 increased the proliferation of myofibroblasts via the MAPK and phosphatidylinositol 3-kinase (PI3K) pathways. In conclusion, stimulation of gastric MMP-7 by elevated plasma gastrin may activate epithelial-mesenchymal signaling pathways regulating myofibroblast function via MAPK and PI3K pathways and contribute to stromal deposition in ECL cell carcinoid tumors.