Recovery Of Mucosal Barrier Function Research Articles

Tu1325 Association Between Common Functional Genetic Polymorphisms and CpG Island Methylation Status in Colonic Mucosa in Ulcerative Colitis

Introduction/Aims: Lubiprostone enhances intestinal fluid secretion by activation of ClC-2, and likely CFTR chloride channels, and is used clinically to treat constipation. It has also been shown to stimulate recovery of mucosal barrier function in ischemic porcine intestine and modify tight junction (TJ) proteins. A defect in barrier function is thought to contribute to both the pathogenesis of inflammatory bowel disease (IBD) and to disease relapse. The aim of this study was to investigate if lubiprostone improves barrier function in colonic biopsies from IBD patients. Methods: Mucosal biopsies were obtained from the sigmoid colon of normal subjects undergoing screening colonoscopy, Crohn's disease (CD) and ulcerative colitis (UC) patients in remission with approval of UCSD IRB. Biopsies were mounted in Ussing chambers, and bathed in Ringer's solution at 37oC. After stabilization for 30 minutes, the tissues were treated with lubiprostone (0.01, 0.1 or 1.0 μM), or vehicle. Paracellular permeability to macromolecules was assessed with the fluorescent marker, FITCdextran (4 kD). Transepithelial electrical resistance (TER) was monitored over 120 minutes. Ion transport responses, measured as short-circuit current (Isc), were recorded for lubiprostone, the cAMP agonist forskolin, and the calcium agonist carbachol (CCh). Statistical analysis was performed using ANOVA or Student's t-test. Results: Lubiprostone increased Isc across human colon in a concentration-dependent manner in all tissues (p<0.05; n=1517). Peak responses to lubiprostone were significantly lower in CD and UC vs. controls (p<0.05; n=15-17). The Isc response to forskolin was similar in controls, CD and UC tissues, while lubiprostone reduced the Isc response to forskolin in a dose-dependent manner. The Isc response to CChwas similar in all three subject groups and was unaffected by lubiprostone pre-treatment. Lubiprostone (1.0 μM) increased the diminished TER in CD biopsies vs. untreated by 8 ± 3 % (n=17, p<0.05) but had no significant effect on UC or normal biopsies. FITC-dextran permeability was not increased in CD or UC vs. controls and was unaffected by lubiprostone. Conclusion: Lubiprostone restored TER in CD biopsies indicating a possible role for lubiprostone as an enhancer of barrier function in CD vs. UC. Moreover, the Isc response to lubiprostone was lower in biopsies from IBD patients vs. normal controls whereas responses to forskolin or CCh were unchanged by disease status. This suggests that lubiprostone could be used to improve barrier function in CD without a risk of excessive fluid secretion. Support provided by Takeda Pharmaceuticals North America, Inc. & NIH DK080506.

The effects of a novel anti‐inflammatory compound (AHI‐805) on cyclooxygenase enzymes and the recovery of ischaemia injured equine jejunumex vivo

Flunixin meglumine is used for treatment of equine colic despite evidence of inhibited recovery of mucosal barrier function following small intestinal ischaemic injury. This study aimed to characterise an alternative treatment (AHI-805) for abdominal pain in the horse. To determine the effect of AHI-805, an aza-thia-benzoazulene derivative, on the cyclooxygenase enzymes and the recovery of mucosal barrier function following ischaemic injury. Effect of AHI-805 on in vitro COX-1 and COX-2 activity was determined by measuring coagulation-induced thromboxane B(2) (TXB(2)) and lipopolysaccharide-stimulated prostaglandin E(2) concentrations in equine whole blood. Horses (n = 6) were anaesthetised and jejunum subjected to ischaemia for 2 h. Control and ischaemia injured mucosa was placed in Ussing chambers and treated with Ringer's solution containing control treatment (DMSO), flunixin meglumine (27 µmol/l), or AHI-805 (27 µmol/l). Transepithelial electrical resistance (TER), mucosal-to-serosal flux of (3) H-mannitol, and bathing solution TXB(2) and prostaglandin E metabolites (PGEM) were measured over a 4 h recovery period. Treatment with AHI-805 had no significant effect on TXB(2) production but significantly inhibited production of PGE(2) at a concentration of 1 µmol/l or greater. TER of flunixin or AHI-805 treated ischaemia-injured jejunum was significantly lower than control treated injured tissue over the recovery period. Mannitol flux and grade of histological damage were significantly increased by ischaemic injury only. There was a significant increase in PGEM and TXB(2) in control tissues over the 240 min recovery period, but not in flunixin or AHI-805 treated tissues. Flunixin meglumine and AHI-805 inhibit recovery of barrier function in ischaemic-injured equine jejunum in vitro through inhibition of the COX enzymes. The novel compound AHI-805 may not be suitable for the treatment of equine colic associated with ischaemic injury.

Effects of the novel anti‐inflammatory drugs robenacoxib and AHI‐805 on recovery of ischemic injured equine jejunum ex vivo

Flunixin is an NSAID administered to horses to ameliorate shock, but which inhibits recovery of barrier function in ischemic‐injured intestine. We examined the effect of robenacoxib, a COX‐2 inhibitor, and AHI‐805, an aza‐thia‐benzoazulene derivative, on recovery of mucosal barrier function. Jejunum was subjected to ischemia for 2 hours in 6 horses. Control and ischemic‐injured mucosa was treated with Ringer's alone (control) or containing 0.27μM flunixin, robenacoxib, or AHI‐805 in Ussing chambers. Trans‐epithelial electrical resistance (TER) and mucosal‐to‐serosal flux of 3H‐mannitol were measured over a 4‐hour period. TXB2 and PGEM in the serosal bathing solution were measured to assess COX‐1 and COX‐2 function. Expression of COX‐1 and ‐2 was determined by immunoblot and histology was performed. Ischemic injury significantly increased 3H‐mannitol flux, epithelial denudation, and COX‐2 expression (P&lt;0.05). TER of ischemic‐injured tissue treated with flunixin or AHI‐805 was significantly lower than control over the recovery period (P&lt;0.05). Robenacoxib did not inhibit recovery of TER. PGEM and TXB2 were significantly decreased by flunixin and AHI‐805 when compared to Ringer's alone or robenacoxib. Robenacoxib permits recovery of ischemic‐injured jejunum judging by indices of barrier function, and this is linked to its ability to allow enhanced PG production.

S1272 Lubiprostone Is Effective and Well Tolerated Through 48 Weeks of Treatment in Adults with Irritable Bowel Syndrome and Constipation

Irritable bowel syndrome (IBS) is a chronic gastrointestinal (GI) disorder consisting of abdominal discomfort/pain, • bloating, and altered bowel habit not explained by structural or biochemical abnormalities. The prevalence of IBS in Western countries is 10-15%. In the USA more than 30 million people suffer from IBS. • – Up to 30% suffer with constipation-predominant IBS (IBS-C). – IBS affects more females than males, at a ratio of 2:1. Individuals with IBS-C experience substantial decrements in the quality of life and their ability to perform activities • of daily living. Lubiprostone, a selective activator of type-2 chloride channels (ClC-2), is approved for the treatment of chronic • idiopathic constipation in adults and for the treatment of IBS-C in adult women. Lubiprostone enhances fluid secretion into the intestinal lumen without altering serum electrolyte levels. • Patients with IBS may exhibit abnormal gut permeability and an associated intestinal inflammatory response. • Lubiprostone stimulates recovery of mucosal barrier function in animal models suggesting a possible mechanism for the clinical improvement observed in patients on this drug.* In 2 previous double-blinded placebo-controlled phase 3 trials in patients with IBS-C, lubiprostone (16 mcg) was • found to be efficacious and well-tolerated for 12 weeks.

Recovery of mucosal barrier function in ischemic porcine ileum and colon is stimulated by a novel agonist of the ClC-2 chloride channel, lubiprostone

Previous studies utilizing an ex vivo porcine model of intestinal ischemic injury demonstrated that prostaglandin (PG)E(2) stimulates repair of mucosal barrier function via a mechanism involving Cl(-) secretion and reductions in paracellular permeability. Further experiments revealed that the signaling mechanism for PGE(2)-induced mucosal recovery was mediated via type-2 Cl(-) channels (ClC-2). Therefore, the objective of the present study was to directly investigate the role of ClC-2 in mucosal repair by evaluating mucosal recovery in ischemia-injured intestinal mucosa treated with the selective ClC-2 agonist lubiprostone. Ischemia-injured porcine ileal mucosa was mounted in Ussing chambers, and short-circuit current (I(sc)) and transepithelial electrical resistance (TER) were measured in response to lubiprostone. Application of 0.01-1 microM lubiprostone to ischemia-injured mucosa induced concentration-dependent increases in TER, with 1 microM lubiprostone stimulating a twofold increase in TER (DeltaTER = 26 Omega.cm(2); P < 0.01). However, lubiprostone (1 microM) stimulated higher elevations in TER despite lower I(sc) responses compared with the nonselective secretory agonist PGE(2) (1 microM). Furthermore, lubiprostone significantly (P < 0.05) reduced mucosal-to-serosal fluxes of (3)H-labeled mannitol to levels comparable to those of normal control tissues and restored occludin localization to tight junctions. Activation of ClC-2 with the selective agonist lubiprostone stimulated elevations in TER and reductions in mannitol flux in ischemia-injured intestine associated with structural changes in tight junctions. Prostones such as lubiprostone may provide a selective and novel pharmacological mechanism of accelerating recovery of acutely injured intestine compared with the nonselective action of prostaglandins such as PGE(2).

Physiological concentrations of bile salts inhibit recovery of ischemic-injured porcine ileum.

We have previously shown rapid in vitro recovery of barrier function in porcine ischemic-injured ileal mucosa, attributable principally to reductions in paracellular permeability. However, these experiments did not take into account the effects of luminal contents, such as bile salts. Therefore, the objective of this study was to evaluate the role of physiological concentrations of deoxycholic acid in recovery of mucosal barrier function. Porcine ileum was subjected to 45 min of ischemia, after which mucosa was mounted in Ussing chambers and exposed to varying concentrations of deoxycholic acid. The ischemic episode resulted in significant reductions in transepithelial electrical resistance (TER), which recovered to control levels of TER within 120 min, associated with significant reductions in mucosal-to-serosal (3)H-labeled mannitol flux. However, treatment of ischemic-injured tissues with 10(-5) M deoxycholic acid significantly inhibited recovery of TER with significant increases in mucosal-to-serosal (3)H-labeled mannitol flux, whereas 10(-6) M deoxycholic acid had no effect. Histological evaluation at 120 min revealed complete restitution regardless of treatment, indicating that the breakdown in barrier function was due to changes in paracellular permeability. Similar effects were noted with the application of 10(-5) M taurodeoxycholic acid, and the effects of deoxycholic acid were reversed with application of the Ca(2+)-mobilizing agent thapsigargin. Deoxycholic acid at physiological concentrations significantly impairs recovery of epithelial barrier function by an effect on paracellular pathways, and these effects appear to be Ca(2+) dependent.

Mitogen-activated protein kinases regulate COX-2 and mucosal recovery in ischemic-injured porcine ileum.

Mitogen-activated protein kinase (MAPK) pathways transduce signals from a diverse array of extracellular stimuli. The three primary MAPK-signaling pathways are the extracellular regulated kinases (ERK1/2), p38 MAPK, and c-Jun NH(2)-terminal kinase (JNK). Previous research in our laboratory has shown that COX-2-elaborated prostanoids participate in recovery of mucosal barrier function in ischemic-injured porcine ileum. Because COX-2 expression is regulated in part by MAPKs, we postulated that MAPK pathways would play an integral role in recovery of injured mucosa. Porcine mucosa was subjected to 45 min of ischemia, after which tissues were mounted in Ussing chambers, and transepithelial electrical resistance (TER) was monitored as an index of recovery of barrier function. Treatment of tissues with the p38 MAPK inhibitor SB-203580 (0.1 mM) or the ERK1/2 inhibitor PD-98059 (0.1 mM) abolished recovery. Western blot analysis revealed that SB-203580 inhibited upregulation of COX-2 that was observed in untreated ischemic-injured mucosa, whereas PD-98059 had no effect on COX-2 expression. Inhibition of TER recovery by SB-203580 or PD-98059 was overcome by administration of exogenous prostaglandin E(2) (1 microM). The JNK inhibitor SP-600125 (0.1 mM) significantly increased TER and resulted in COX-2 upregulation. COX-2 expression appears to be positively and negatively regulated by the p38 MAPK and the JNK pathways, respectively. Alternatively, ERK1/2 appear to be involved in COX-2-independent reparative events that remain to be defined.

Neutrophils increase paracellular permeability of restituted ischemic-injured porcine ileum

Background. We have previously shown minimal evidence of neutrophil infiltration during early reperfusion of porcine ischemic ileum. However, we noted marked neutrophil infiltration 6 to 18 hours after ischemia during mucosal repair. We postulated such neutrophil infiltration would disrupt restituting epithelium. Methods. Pigs were pretreated with anti-CD11/CD18 monoclonal antibody, superoxide dismutase-polyethylene glycol, or saline solution before inducing 1 hour of ischemia. Pigs recovered for up to 18 hours, after which mucosal repair was assessed. Results. One hour of ischemia induced loss of 19 ± 7% of the villous epithelial surface area. Epithelial restitution covered the mucosal defect within 2 hours, although full recovery of mucosal barrier function required 6 hours. By 18 hours, a significant decrease in transepithelial electrical resistance and increase in transmucosal mannitol flux was noted despite the continued presence of complete epithelial coverage. Accumulation of neutrophils within restituting epithelium was noted on histologic examination, associated with electron-microscopic evidence of widened paracellular spaces. Pretreatment with anti-CD11/CD18 monoclonal antibody and superoxide dismutase-polyethylene glycol significantly reduced neutrophil infiltration and normalized transepithelial electrical resistance and mannitol fluxes. Conclusions. Mucosal inflammation during epithelial repair resulted in increased paracellular permeability as neutrophils traversed restituted epithelium. Blocking neutrophil adhesion or scavenging superoxide prevented mucosal dysfunction in recovering tissue. (Surgery 2002;132:461-70.)

Energy dependence of restitution in the gastric mucosa.

Rapid epithelial repair (restitution) after injury is required to maintain barrier function of the gastrointestinal mucosa and skin and is thought to be a highly ATP-dependent process that would be inhibited under hypoxic conditions. However, little is known about the metabolic pathways required for restitution. Thus, this study was undertaken to evaluate, in vitro, the role of oxidative respiration and glycolysis in restitution after injury. To this end, restitution of the bullfrog gastric mucosa was evaluated under the following conditions: 1) blockade of mitochondrial respiration; 2) blockade of glycolysis; or 3) absence of glucose. The extent of mucosal repair after injury was evaluated by electrophysiology and morphology. Cell migration, repolarization, and the formation of tight junctions after injury occurred during blockade of mitochondrial respiration, whereas the recovery of mucosal barrier function did not. In contrast, glycolytic inhibition completely blocked all aspects of restitution by inhibiting the migration of surface epithelial cells. Restitution occurred in tissues incubated with glucose-free solutions, suggesting that cells contain sufficient glucose (glycogen) to drive glycolysis for many hours. Our results demonstrate that the glycolytic pathway is essential for restitution after injury in the bullfrog gastric mucosa and that all but complete repair of barrier function occurs in the absence of mitochondrial respiration.

Synergistic signaling via cAMP and CA 2+1-coupled EP receptors is required for PGE 2-triggered recovery of intestinal barrier function

We have recently shown that PGI2 and PG~ have a synergistic role in rescuing epithelial barrier function in porcine ischemic-injured ileum. While it appears that PGI2 signals via cholinergic nerve-induced increases in Ca2+ ;, and PGE2 signals via receptor-mediated release of cAMP, the precise nature of PGE2 receptor interactions that mediate recovery of mucosal barrier function is not known. PGE2 may induce increases in cAMP via EP2 or EP4 receptors and increases in Ca2+; via EP I receptors. However, PGErEP3 receptor signaling pathways are more complex, and may result in increases or decreases in cAMP or increases in Ca2+i depending upon the splice variant of the EP3 receptor present. The objective of the present study was to define the PGE2 signaling pathways involved in recovery of mucosal barrier function. Porcine ileum was subjected to 45-minutes of complete ischemia in vivo, after which the mucosa was stripped and mounted in Ussing chambers. Transepithelial resistance (R) was serially monitored over a 4-hour period as an indicator of mucosal recovery. All tissues were pre-treated with indomethacin (5 x 1O-6M). Ischemia resulted in 50% reductions in R (26::':2.70ecm2) compared to control tissues (51::':3.20ecm2). Treatment with the EP,IEP2IEPi EP4 agonist 16,16-dimethyl PG~ (PGE2, 1O6M) triggered 2-fold elevations in R (51::':4.20ecm2)within I hour, whereas the EP2IEP4 agonist deoxyPGE, (10-6M) or the EP,IEP3 agonist sulprostone (10-6M) had no effect. However, a combination of deoxyPGE, and sulprostone simulated the action of PGE2.cAMP was significantly elevated in tissues treated with PGE2 (23.2::':2.2 vs. 14.9::':2.2 pmol/ml for control) and deoxyPGErf sulprostone (49.1::':4.2 pmol/ml), but not with deoxyPGE, or sulprostone alone. Because of the potential for EP, and EP3 receptors to trig~er elevations in Ca2+;, we applied deoxyPGE, in combination with the Ca + ionophore A23187. Treatment with A23187 (1Oand deoxyl'Gfi, resulted in elevations in R similar in magnitude to those of PGE 2 (55::':6.40ecm2), whereas treatment with A23187 alone triggered significant but less marked elev~tions in R (45::':4.60ecm.j,. Pre-treatment with the EP I receptor antagonists SC-19220 (106 10 M) or AH-6809(l0sM)had no effect on PGE2 or sulprostone/deoxyPGE,-induced elevations in R. These studies suggest PGE2 signals recovery of R in ischemic-injured porcine ileum via complex si~a1ing pathways requiring both cAMP (via EP2 or EP4 receptors) and Ca +, (most likely via EP3 receptors).