Mucosal Restitution Research Articles

THE FUNCTION OF THE CYSTATHIONINE-GAMMA-LYASE/HYDROGEN SULFIDE AXIS IN THE PATHOGENESIS OF ULCERATIVE COLITIS

Abstract INTRODUCTION Current combination therapies in ulcerative colitis (UC) target different inflammatory cascades, including tumor necrosis factor-alpha (TNF-alpha) signaling. However, most patients do not achieve long-term remission, suggesting that the molecular pathophysiology of UC is still poorly understood. Endogenously produced hydrogen sulfide (H2S) has emerged as a critical physiological mediator of gastrointestinal homeostasis, limiting mucosal inflammation and promoting tissue healing in response to epithelial injury. Inhibition of cystathionine-gamma-lyase (CSE)-dependent H2S production in animal models of UC has been shown to exacerbate colitis and delay tissue repair. Our research group has strong evidence revealing that the CSE/H2S axis regulates TNF-alpha signaling in colonic epithelial cells. Therefore, we hypothesize that decreased CSE/H2S pathway activity in colonic mucosa cells leads to dysregulation of TNF-alpha signaling and uncontrolled inflammatory/immune responses with aberrant wound repair mechanisms contributing to the pathogenesis of UC. RESULTS We found a significant decrease in CSE mRNA and protein expression in colonic biopsies from patients with UC. Using a preclinical animal model of epithelium injury-induced chronic colitis (DSS), we confirmed a sustained downregulation of CSE, as well as upregulation of TNF-alpha and tumor necrosis receptor 2 expressions in colonic mucosa samples. Treatment with the slow-releasing H2S-donor GYY4137 significantly reduced the severity of DSS-induced inflammation and improved mucosal integrity and restitution responses. In epithelial cell and colonoid cultures, the inhibition of CSE-dependent H2S synthesis decreased the activation of extracellular-signal-regulated-kinase-1/2 (ERK1/2), increased IL-6 and TNF-alpha mRNA levels and suppressed basic and TNF-alpha–mediated colonocyte migration. CONCLUSION These studies demonstrate that CSE regulates mucosal TNF-alpha signaling, pro- and anti-inflammatory responses as well as colonic epithelial repair. Adding the H2S donor GYY4137 reduces inflammation, suggesting the restoration of normal TNF-alpha signaling function. Acknowledgments: KM is supported by the DoD Career Development Award (W81XWH2010641) and ACS Research Scholar Grant (RSG–21–027–01–CSM).

Enteric glial cell network function is required for epithelial barrier restitution following intestinal ischemic injury in the early postnatal period.

Ischemic damage to the intestinal epithelial barrier, such as in necrotizing enterocolitis or small intestinal volvulus, is associated with higher mortality rates in younger patients. We have recently reported a powerful pig model to investigate these age-dependent outcomes in which mucosal barrier restitution is strikingly absent in neonates but can be rescued by direct application of homogenized mucosa from older, juvenile pigs by a yet-undefined mechanism. Within the mucosa, a postnatally developing network of enteric glial cells (EGCs) is gaining recognition as a key regulator of the mucosal barrier. Therefore, we hypothesized that the developing EGC network may play an important role in coordinating intestinal barrier repair in neonates. Neonatal and juvenile jejunal mucosa recovering from surgically induced intestinal ischemia was visualized by scanning electron microscopy and the transcriptomic phenotypes were assessed by bulk RNA sequencing. EGC network density and glial activity were examined by Gene Set Enrichment Analysis, three-dimensional (3-D) volume imaging, and Western blot and its function in regulating epithelial restitution was assessed ex vivo in Ussing chamber using the glia-specific inhibitor fluoroacetate (FA), and in vitro by coculture assay. Here we refine and elaborate our translational model, confirming a neonatal phenotype characterized by a complete lack of coordinated reparative signaling in the mucosal microenvironment. Furthermore, we report important evidence that the subepithelial EGC network changes significantly over the early postnatal period and demonstrate that the proximity of a specific functional population of EGC to wounded intestinal epithelium contributes to intestinal barrier restitution following ischemic injury.NEW & NOTEWORTHY This study refines a powerful translational pig model, defining an age-dependent relationship between enteric glia and the intestinal epithelium during intestinal ischemic injury and confirming an important role for enteric glial cell (EGC) activity in driving mucosal barrier restitution. This study suggests that targeting the enteric glial network could lead to novel interventions to improve recovery from intestinal injury in neonatal patients.

Curcumin promotes renewal of intestinal epithelium by miR-195-3p

Ethnopharmacological relevanceTurmeric (Curcuma longa) has been used to treat gastrointestinal disorders in the Indian Ayurvedic medical system. According to the theory behind traditional Chinese medicine, turmeric can be distributed in the spleen meridian, for which it has been used as a digestive aid. Curcumin (Cur), a natural polyphenol compound originally derived from turmeric, has anti-inflammatory activity and can assist in treating inflammatory bowel disease. Aims of the studyTo investigate curcumin's protective effects on intestinal epithelium and explore the underlying miR-195-3p-related mechanisms. Materials and methodsThe miR-195-3p mimics were used to over-express miR-195-3p. The in vitro effects of Cur and miR-195-3p on the intestine were shown utilizing intestinal cryptlike epithelial cell line-6 (IEC-6) cells. By fasting for 48 h, an intestinal mucosal atrophy model of SD rats was created in vivo. Cur (25 or 50 mg/kg) was assessed for its protective effect on intestinal epithelium. Glycyrrhetinic acid (GA) with an intestinal protective effect reported in our previous research was adopted as a positive drug for the in vivo and in vitro bioactivity evaluation since there is no universally positive drug for either intestinal mucosal restitution or miR-195-3p modulation. ResultsCur protects the intestinal epithelium and promotes its repair after injury via down-regulating miR-195-3p. In vitro experiments showed that Cur inhibited the apoptosis of IEC-6 cells, stimulated their growth, and down-regulated the level of miR-195-3p in cells. When miR-195-3p was overexpressed, the viability of IEC-6 cells decreased while the apoptosis rate increased. All the above detrimental effects were alleviated after curcumin intervention. Moreover, Cur reversed the effect of miR-195-3p on its downstream occludin. In vivo, results showed that 48-h fasting impaired the integrity of the small intestinal mucosa (abnormal crypt structure and reduced goblet cell number), which was ameliorated by Cur treatment. In addition, the Cur treatment reversed both the increased expression level of miR-195-3p and decreased levels of ki-67 and occludin caused by fasting. ConclusionsCur could promote the proliferation and repair after injury of the intestinal mucosa by down-regulating miR-195-3p.

24 Intestinal Function and Integrity in Health and Disease: Current Knowledge in Swine

Abstract Optimal lean tissue accretion is contingent on the ability of the pig to efficiently establish and maintain host defenses, and to digest and absorb nutrients and water. Epithelial cells in the mucosa of the gastrointestinal tract interconnect with each other by tight junctions, adherens junctions, and desmosomes, forming a complete intestinal barrier. Of the secretory cells in this epithelial barrier, mucus secreting goblet cells and hormone producing enteroendocrine cells can be found interspersed, while Paneth cells lie in the base of the crypts and secrete antimicrobial proteins/peptides into the mucus layer. In the small intestine, enterocytes lining the villi allow for efficient nutrient transport. Maintenance of the epithelial barrier and its function is dependent on intestinal stem cells residing at the crypt base. Two major signaling pathways controlling intestinal stem cell proliferation and differentiation are the β-catenin/Wnt and Notch signaling pathways. Thus, achieving and maintaining optimal nutrient absorptive capacity requires an intricate balance amongst β-catenin/Wnt and Notch signaling pathways, wherein dysregulation of either can result in inefficiency of nutrient absorption. Weaning transition, off-feed events, hypophagia, and enteric and respiratory diseases can all compromise the structural and secretory barrier of the gastrointestinal tract mucosa via changes in tight and adherens junction proteins and mucin production. As a result, these stressors can increase the incidence of gastric ulcers, villous atrophy, and colitis in pigs. Depending on the severity and duration of the mucosal damage, repair and restitution occurs due to increased stem cell proliferation, migration, and repolarization. Thus, efficiency of digestive and absorptive function depends on several factors, including the types of epithelial cells present and their functionality, the rate of cell renewal, and epithelial surface area. This paper will discuss what we know about the regulation and repair of the mucosal epithelium in growing pigs during health, stress, and disease.

Total ginsenosides promotes intestinal epithelial proliferation via affecting polyamine-mediated HuR on post-transcriptional control

Ginseng, as a functional food, is widely used worldwide because of its various benefits. This study aims to elucidate the potential effects of total ginsenosides (TG) in intestinal mucosal restitution. Establishing IEC-6 cell proliferation inhibition model by culturing with 5 mmol/L DL-α-difluoromethylornithine (DFMO, a polyamine inhibitor) for 48 h. The intestinal injury mice model was induced by 3% Dextran sodium sulfate (DSS) for 7 d. TG reversed the cell growth inhibition, regulated cell cycle and the decrease in c-Myc protein expression caused by DFMO, without affecting mRNA expression; TG increased translation efficiency and half-life of c-Myc mRNA, suggesting that TG promoted cell proliferation through affecting translation of c-Myc. Furthermore, TG could prevent the decrease of human antigen R (HuR)/c-Myc mRNA, p-HuR and p-Chk2 expression caused by DFMO, as well as reduce the expression of HuR in cytoplasm while increasing the expression in nucleus, indicating that TG influenced post-transcriptional control of c-Myc through polyamine-mediated HuR pathway for intestinal mucosal restitution. Panax ginseng aqueous extract (PGE) prevented intestinal mucosal injury induced by DSS(improved clinical symptoms and restored colon integrity) through enhancing HuR and c-Myc levels. Our results may provide a scientific basis for ginseng as a potential functional food ingredient and assistance therapeutic agent to prevent intestinal injury.

PDGFA-associated protein 1 is a novel target of c-Myc and contributes to colorectal cancer initiation and progression.

BackgroundThe mechanism underlying colorectal cancer (CRC) initiation and progression remains elusive, and overall survival is far from satisfactory. Previous studies have shown that PDGFA‐associated protein 1 (PDAP1) is upregulated in several cancers including CRC. Here, we aimed to identify the cause and consequence of PDAP1 dysregulation in CRC and evaluate its role as a potential therapeutic target.MethodsMulti‐omics data analysis was performed to identify potential key players in CRC initiation and progression. Immunohistochemistry (IHC) staining was applied to determine the expression pattern of PDAP1 in CRC tissues. Pdap1 conditional knockout mice were used to establish colitis and CRC mouse models. RNA sequencing, a phosphoprotein antibody array, western blotting, histological analysis, 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay, and interactome analysis were applied to identify the underlying mechanisms of PDAP1. A human patient‐derived xenograft (PDX) model was used to assess the potential of PDAP1 as a therapeutic target.ResultsPDAP1 was identified as a potential key player in CRC development using multi‐omics data analysis. PDAP1 was overexpressed in CRC cells and correlated with reduced overall survival. Further investigation showed that PDAP1 was critical for the regulation of cell proliferation, migration, invasion, and metastasis. Significantly, depletion of Pdap1 in intestinal epithelial cells impaired mucosal restitution in dextran sulfate sodium salt‐induced colitis and inhibited tumor initiation and growth in colitis‐associated cancers. Mechanistic studies showed that c‐Myc directly transactivated PDAP1, which contributed to the high PDAP1 expression in CRC cells. PDAP1 interacted with the juxtamembrane domain of epidermal growth factor receptor (EGFR) and facilitated EGFR‐mitogen‐activated protein kinase (MAPK) signaling activation, which resulted in FOS‐related antigen 1 (FRA‐1) expression, thereby facilitating CRC progression. Notably, silencing of PDAP1 could hinder the growth of patient‐derived xenografts that sustain high PDAP1 levels.ConclusionsPDAP1 facilitates mucosal restitution and carcinogenesis in colitis‐associated cancer. c‐Myc‐driven upregulation of PDAP1 promotes proliferation, migration, invasion, and metastasis of CRC cells via the EGFR‐MAPK‐FRA‐1 signaling axis. These findings indicated that PDAP1 inhibition is warranted for CRC patients with PDAP1 overexpression.

Polysaccharides from Panax ginseng promote intestinal epithelial cell migration through affecting the Ca2+ related regulators

Background and aimPanax ginseng, a key herbal medicine of replenishing Qi and tonifying Spleen, is widely used in the treatment of gastrointestinal diseases in East Asia. In this study, we aim to investigate the potential effects and mechanisms of polysaccharides from P. ginseng (PGP) on intestinal mucosal restitution which is one of the crucial repair modalities during the recovery of mucosal injury controlled by the Ca2+ signaling. MethodsRat model of intestinal mucosal injury was induced by indomethacin. The fractional cell migration was carried out by immunohistochemistry staining with BrdU. The morphological observations on intestinal mucosal injury were also performed. Intestinal epithelial cell (IEC-6) migration in vitro was conducted by scratch method. Western-blot was adopted to determine the expressions of PLC-γ1, Rac1, TRPC1, RhoA and Cav-1. Immunoprecipitation was used to evaluate the levels of Rac1/PLC-γ1, RhoA/TRPC1 and Cav-1/TRPC1. ResultsThe results showed that PGP effectively reduced the assessment of intestinal mucosal injury, reversed the inhibition of epithelial cell migration induced by Indomethacin, and increased the level of Ca2+ in intestinal mucosa in vivo. Moreover, PGP dramatically promoted IEC-6 cell migration, the expression of Ca2+ regulators (PLC-γ1, Rac1, TRPC1, Cav-1 and RhoA) as well as protein complexes (Rac1/PLC-γ1, Cav-1/TRPC1 and RhoA/TRPC1) in vitro. ConclusionPGP increases the Ca2+ content in intestinal mucosa partly through controlling the regulators of Ca2+ mobilization, subsequently promotes intestinal epithelial cell migration, and then prevents intestinal mucosal injury induced by indomethacin.

CD147 receptor is essential for TFF3-mediated signaling regulating colorectal cancer progression

Major gaps in understanding the molecular mechanisms of colorectal cancer (CRC) progression and intestinal mucosal repair have hampered therapeutic development for gastrointestinal disorders. Trefoil factor 3 (TFF3) has been reported to be involved in CRC progression and intestinal mucosal repair; however, how TFF3 drives tumors to become more aggressive or metastatic and how TFF3 promotes intestinal mucosal repair are still poorly understood. Here, we found that the upregulated TFF3 in CRC predicted a worse overall survival rate. TFF3 deficiency impaired mucosal restitution and adenocarcinogenesis. CD147, a membrane protein, was identified as a binding partner for TFF3. Via binding to CD147, TFF3 enhanced CD147-CD44s interaction, resulting in signal transducer and activator of transcription 3 (STAT3) activation and prostaglandin G/H synthase 2 (PTGS2) expression, which were indispensable for TFF3-induced migration, proliferation, and invasion. PTGS2-derived PGE2 bound to prostaglandin E2 receptor EP4 subtype (PTGER4) and contributed to TFF3-stimulated CRC progression. Solution NMR studies of the TFF3-CD147 interaction revealed the key residues critical for TFF3 binding and the induction of PTGS2 expression. The ability of TFF3 to enhance mucosal restitution was weakened by a PTGS2 inhibitor. Blockade of TFF3-CD147 signaling using competitive inhibitory antibodies or a PTGS2 inhibitor reduced CRC lung metastasis in mice. Our findings bring strong evidence that CD147 is a novel receptor for TFF3 and PTGS2 signaling is critical for TFF3-induced mucosal restitution and CRC progression, which widens and deepens the understanding of the molecular function of trefoil factors.

The RNA-binding protein HuR regulates intestinal epithelial restitution by modulating Caveolin-1 gene expression.

The integrity of the intestinal mucosal barrier protects hosts against pathological conditions. Early mucosal restitution after wounding refers to epithelial cell migration into a defect. The RNA-binding protein HuR plays an important role in the posttranscriptional regulation of gene expression and is involved in many aspects of cellular physiology. In the present study, we investigated the role of HuR in the regulation of cell migration through the posttranscriptional regulation of Caveolin-1 (Cav-1). Online software was used to identify Cav-1 mRNA as a potential target of HuR. The interaction of HuR with Cav-1 mRNA was investigated via ribonucleoprotein immunoprecipitation (RNP IP) assays and biotin pulldown analysis. HuR was found to bind specifically to the Cav-1 3'-UTR rather than the coding region or 5'-UTR. Transfection of cells with siHuR decreased both HuR protein levels and Cav-1 protein levels; conversely, ectopic overexpression of HuR via infection of cells with an adenoviral vector containing HuR cDNA (AdHuR) increased Cav-1 protein levels without disturbing Cav-1 mRNA levels. Thus, HuR enhanced Cav-1 expression in vitro by stimulating Cav-1 translation. Intestinal epithelium-specific HuR knockout in mice decreased Cav-1 protein levels without changing Cav-1 mRNA levels, consistent with the in vitro results. Decreasing the levels of HuR via siHuR transfection inhibited early epithelial repair, but this effect was reversed by ectopic overexpression of GFP-tagged Cav-1. These results indicate that posttranscriptional regulation of Cav-1 gene expression by HuR plays a critical role in the regulation of rapid epithelial repair after wounding.

Histologic changes in early colonic anastomotic healing using autologous platelet-rich fibrin matrix

Anastomotic leakage represents a devastating post-surgical event with surgeon- and patient-related causes. The early lag phase of healing (day 0–4) is a time of increased vulnerability. The current role of tissue sealants as anastomotic adjuncts remains unclear, with controversial results reported. Platelet-rich plasma provides a reliable source of growth factors supporting wound healing. We aimed to assess the impact of a novel autologous platelet-rich fibrin (PRF) agent (Obsidian-ASG® matrix; GergMed Medizintechnik, Baiern, Germany) on colonic anastomotic healing in a porcine model. Left-sided colonic anastomoses were constructed in 16 pigs with reinforcement using the Obsidian-ASG® matrix in 12 animals. Animals were sacrificed on days 0, 4, 10 and 30, with histologic assessment of the anastomotic line along with evaluation of fibrotic tissue maturation, foreign body reactivity and mucin production. Collagen maturity and immunohistochemical changes of angiogenesis and infiltrating macrophage profiles were determined as well. There was no observable effect in matrix-treated animals on epithelial line thickness, granulation tissue formation, foreign body reactivity or mucosal restitution. Matrix-treated animals showed higher mucin production on day 4 accompanied by a more mature collagen and a greater degree of early angiogenic response with a higher infiltration density of M2 macrophages. Additionally, anastomotic burst pressure in matrix-treated animals was higher on day 0, 4, 10 and 30 (100, 100, 210, 160 vs. 40, 30, 60, 20 mm Hg, respectively). Obsidian-ASG® matrix may have beneficial effects on anastomotic healing by shifting the inflammatory response towards a mature and more stable collagen phenotype in the early phase.

Effect of polysaccharides from Sijunzi decoction on Ca2+ related regulators during intestinal mucosal restitution

BackgroundSijunzi decoction, a representative Chinese herbal formula of reinforcing Qi strengthening spleen, has been widely used for treating patients with gastrointestinal diseases and spleen-deficiency syndrome, however, the exact effects and mechanisms are remained unclear. Hypothesis/purposeThe migration of intestinal epithelial (IEC-6) cells has been demonstrated to be one of the major repair modalities during the healing of mucosal wounds. Ca2+ is the key factor in regulating cell migration. Thus, this study aimed to elucidate the potential effects and mechanisms of polysaccharides (SJZDP) extracted from Sijunzi decoction in intestinal mucosal restitution. MethodCell migration was detected by scratch method with micropipette tip. Western blotting was adopted to evaluate the expression of STIM1 and STIM2 proteins. Immunofluorescence was carried out to assess the translocation of STIM1 protein. Immunoprecipitation was used to determine the levels of STIM1/TRPC1 and STIM1/STIM2 complexes. A indomethacin-induced intestinal mucosal injury rat model was applied. The content of polyamines in intestinal mucosa was detected by high-performance liquid chromatography. The morphological changes in the intestinal mucosa were observed at the end of animal experiment. ResultsThe results showed that treatment with SJZDP significantly promoted cell migration, enhanced the level of STIM1 protein and STIM1/TRPC1 complexes, reduced the level of STIM2 protein and STIM1/STIM2 complexes. Further more, SJZDP exposure promoted the translocation of STIM1 to the plasma membrane. In vivo experiment results, the administration of SJZDP effectively reduced intestinal mucosal injury. ConclusionThese results revealed that SJZDP promotes intestinal epithelial restitution after wounding, presumably by regulating cellular levels of STIM1 and STIM2 differentially, stimulating the translocation of STIM1, and inducing STIM1/TRPC1 association as well as decreasing the level of STIM1/STIM2.

Loss of trefoil factor 1 inhibits biliary regeneration but accelerates the hepatic differentiation of progenitor cells in mice

Although the regeneration of the adult liver depends on hepatic progenitor cells (HPCs), many uncertainties regarding hepatic regeneration in the injured liver remain. Trefoil factor family 1 (TFF1), a secretory protein predominantly expressed in the gastrointestinal tract, is responsible for mucosal restitution. Here, we investigated the role of TFF1 in liver regeneration using a mouse model of hepatic injury (choline-deficient ethionine-supplemented diet and carbon tetrachloride administration) and genetically engineered mice (TFF1 knockout (TFF1−/−)). Immunohistochemistry analysis of human liver samples revealed TFF1 expression in the hepatocytes close to ductular reaction and the regenerating biliary epithelium in injured liver. The number of cytokeratin 19 (CK19)-positive bile ducts was significantly decreased in the TFF1−/− mice after liver injury. Notch pathway in the TFF1−/− mice was also downregulated. HPCs in the control mice differentiated into biliary cells (CK19+/SRY HMG box 9 (SOX9)+) more frequently. In contrast, HPCs in the TFF1−/− mice more frequently differentiated into a hepatic lineage (alpha fetoprotein+/SOX9+) after acute liver damage. Hepatocyte proliferation was upregulated, and the liver weight was increased in TFF1−/− mice in response to chronic liver damage. Thus, TFF1 is responsible for liver regeneration after liver injury by promoting HPC differentiation into a biliary lineage and inhibiting HPC differentiation into a hepatic lineage.

β-PIX plays an important role in regulation of intestinal epithelial restitution by interacting with GIT1 and Rac1 after wounding.

Early gut mucosal restitution is a process by which intestinal epithelial cells (IECs) migrate over the wounded area, and its defective regulation occurs commonly in various critical pathological conditions. This rapid reepithelialization is mediated by different activating small GTP-binding proteins, but the exact mechanism underlying this process remains largely unknown. Recently, it has been reported that interaction between p21-activated kinase-interacting exchange factor (β-PIX) and G protein-coupled receptor kinase-interacting protein 1 (GIT1) activates small GTPases and plays an important role in the regulation of cell motility. Here, we show that induced association of β-PIX with GIT1 is essential for the stimulation of IEC migration after wounding by activating Rac1. Levels of β-PIX and GIT1 proteins and their association in differentiated IECs (line of IEC-Cdx2L1) were much higher than those observed in undifferentiated IECs (line of IEC-6), which was associated with an increase in IEC migration after wounding. Decreased levels of endogenous β-PIX by its gene-silencing destabilized β-PIX/GIT1 complexes, repressed Rac1 activity and inhibited cell migration over the wounded area. In contrast, ectopic overexpression of β-PIX increased the levels of β-PIX/GIT1 complexes, stimulated Rac1 activity, and enhanced intestinal epithelial restitution. Increased levels of cellular polyamines also stimulated β-PIX/GIT1 association, increased Rac1 activity, and promoted the epithelial restitution. Moreover, polyamine depletion decreased cellular abundances of β-PIX/GIT1 complex and repressed IEC migration after wounding, which was rescued by ectopic overexpression of β-PIX or GIT1. These results indicate that β-PIX/GIT1/Rac1 association is necessary for stimulation of IEC migration after wounding and that this signaling pathway is tightly regulated by cellular polyamines. NEW & NOTEWORTHY Our current study demonstrates that induced association of β-PIX with GIT1 is essential for the stimulation of intestinal epithelial restitution by activating Rac1, and this signaling pathway is tightly regulated by cellular polyamines.

Suppression of epithelial restitution using an inhibitor against Rho-associated coiled-coil containing protein kinase aggravates colitis through reduced epithelial expression of A-kinase anchor protein 13

In the gastrointestinal tract, the immediate healing response to mucosal damage is critical to sustain mucosal homeostasis. The migration of surrounding epithelial cells to cover the denuded area without proliferation is termed restitution, followed by early reparation of the damage. In this study, we determined the role of A-kinase anchor protein 13 (AKAP13) in mice with dextran sulphate sodium (DSS)-induced colitis upon mucosal injury and restitution, and investigated whether inhibition of Rho-associated coiled-coil containing protein kinase (ROCK), downstream effector of AKAP13, affects these mucosal responses. BALB/c mice were challenged with 4% or 2% DSS in their drinking water for up to 8 or 16days, respectively. During this period, mice received subcutaneous injections of fasudil hydrochloride hydrate (FH, 10mg/kg, twice per day), an inhibitor of phosphorylation of ROCK. In immunohistochemistry, AKAP13 was highly expressed in the mucosal epithelium prior to DSS-induced mucosal injury, and also expressed in ulcer-covering non-proliferative epithelium, which corresponded to restituted epithelial cells. Coadministration of FH increased serum amyloid A levels and histopathological scores for mucosal injury, as compared with the DSS group. The effects were associated with a decrease in gene expression of Akap13 in the mucosal tissue and the inhibition of restitution rata (the length of restituted epithelial cells per ulcer). These results suggested that AKAP13 and ROCK are involved in mucosal response at early injury and restitution during healing in DSS-induced colitis in mice.

Tu1898 - Steroid Therapy for Inflammatory Bowel Diseases (IBD) Reduces Intestinal Stem Cell (ISC) Activation and Proliferation Required for Mucosal Restitution

Tu1898 - Steroid Therapy for Inflammatory Bowel Diseases (IBD) Reduces Intestinal Stem Cell (ISC) Activation and Proliferation Required for Mucosal Restitution

Acute Infectious Gastroenteritis Potentiates a Crohn's Disease Pathobiont to Fuel Ongoing Inflammation in the Post-Infectious Period.

Crohn’s disease (CD) is a chronic inflammatory condition of diverse etiology. Exposure to foodborne pathogens causing acute gastroenteritis produces a long-term risk of CD well into the post-infectious period but the mechanistic basis for this ongoing relationship to disease onset is unknown. We developed two novel models to study the comorbidity of acute gastroenteritis caused by Salmonella Typhimurium or Citrobacter rodentium in mice colonized with adherent-invasive Escherichia coli (AIEC), a bacterial pathobiont linked to CD. Here, we show that disease activity in the post-infectious period after gastroenteritis is driven by the tissue-associated expansion of the resident AIEC pathobiont, with an attendant increase in immunopathology, barrier defects, and delays in mucosal restitution following pathogen clearance. These features required AIEC resistance to host defense peptides and a fulminant inflammatory response to the enteric pathogen. Our results suggest that individuals colonized by AIEC at the time of acute infectious gastroenteritis may be at greater risk for CD onset. Importantly, our data identify AIEC as a tractable disease modifier, a finding that could be exploited in the development of therapeutic interventions following infectious gastroenteritis in at-risk individuals.

Mechanisms Involved in the Development of the Chronic Gastrointestinal Syndrome in Nonhuman Primates after Total-Body Irradiation with Bone Marrow Shielding.

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K+, or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Regenerating Reputations: Are Wnt and Myc the Good Guys After All?

Inflammatory bowel disease (IBD) is one of the biggest challenges facing gastroenterologists today, due to the lack of efficacy of many of its treatments, its widespread morbidities, and its increasing incidence [1]. One of the etiological factors thought to contribute to IBD pathogenesis is diminished intestinal barrier function, which at least in theory permits luminal antigens such as bacterial products to enter the submucosa [2]. Hence, a major unmet need in IBD treatment is a strategy to reverse barrier breaches by directly promoting mucosal restitution and repair. While the importance of this endpoint has been widely acknowledged [3], no IBD therapies currently in use directly target regeneration of the intestinal or colonic epithelium. Rather, it is currently a case of ‘‘Mucosa, heal thyself!’’ in which repair is expected to occur secondarily to the blockade of inflammation. Nevertheless, a wounded mucosa and its attendant disrupted barrier are thought to contribute to ongoing inflammation. Thus, directly bolstering mucosal regeneration may provide an improved alternative or adjunct approach to IBD treatment. In this issue of Digestive Diseases and Sciences, Raup-Konsavage and colleagues report proof-of-concept data supporting pharmacologically targeting glycogen synthase kinase (GSK)-3b to promote intestinal repair and regeneration through a c-Myc-dependent pathway [4]. The Wnt signaling pathway and the Myc transcription factor are central regulators of cell division that for years have been theoretical clinical targets. They have typically been investigated as targets for inhibition, due to the strongly oncogenic effects of constitutively activating mutants. However, an emerging literature suggests that harnessing these pathways’ ability to promote cell division could be an effective therapeutic approach for the treatment of injury and inflammation, including in the gut. The Yochum laboratory has previously characterized a mouse with a deletion in the 30 Wnt responsive DNA element of the Myc gene [5]. This mouse displayed a moderate increase in Myc protein expression, heightened intestinal crypt proliferation, and accelerated recovery from dextran sulfate sodium (DSS)-induced experimental colitis. Thus, in the current study, they tested whether indirectly inducing Myc by pharmacologically activating Wnt signaling with LiCl promotes healing. The data show that LiCl does indeed stimulate recovery when given to mice with established acute DSS colitis, and that this protection is sensitive to pharmacological Myc inhibition. The authors also demonstrated direct effects of LiCl and Myc on epithelial cells in vitro by observing lithium-induced accelerated scratch assay restitution, which was reversed by the Myc inhibitor. They consequently outline a surprisingly specific pathway driving recovery from an inflammatory insult in the gut, using a drug related to compounds already widely used in human patients with psychiatric disorders, such as Li2CO3. This study is distinct from previous experiments with lithium or Myc activation in mouse colitis models in that the investigators gave lithium therapeutically rather than prophylactically. The therapeutic approach is an important & Mark R. Frey mfrey@usc.edu

RhoA enhances store-operated Ca2+ entry and intestinal epithelial restitution by interacting with TRPC1 after wounding.

Early mucosal restitution occurs as a consequence of epithelial cell migration to resealing of superficial wounds after injury. Our previous studies show that canonical transient receptor potential-1 (TRPC1) functions as a store-operated Ca(2+) channel (SOC) in intestinal epithelial cells (IECs) and plays an important role in early epithelial restitution by increasing Ca(2+) influx. Here we further reported that RhoA, a small GTP-binding protein, interacts with and regulates TRPC1, thus enhancing SOC-mediated Ca(2+) entry (SOCE) and epithelial restitution after wounding. RhoA physically associated with TRPC1 and formed the RhoA/TRPC1 complexes, and this interaction increased in stable TRPC1-transfected IEC-6 cells (IEC-TRPC1). Inactivation of RhoA by treating IEC-TRPC1 cells with exoenzyme C3 transferase (C3) or ectopic expression of dominant negative RhoA (DNMRhoA) reduced RhoA/TRPC1 complexes and inhibited Ca(2+) influx after store depletion, which was paralleled by an inhibition of cell migration over the wounded area. In contrast, ectopic expression of wild-type (WT)-RhoA increased the levels of RhoA/TRPC1 complexes, induced Ca(2+) influx through activation of SOCE, and promoted cell migration after wounding. TRPC1 silencing by transfecting stable WT RhoA-transfected cells with siRNA targeting TRPC1 (siTRPC1) reduced SOCE and repressed epithelial restitution. Moreover, ectopic overexpression of WT-RhoA in polyamine-deficient cells rescued the inhibition of Ca(2+) influx and cell migration induced by polyamine depletion. These findings indicate that RhoA interacts with and activates TRPC1 and thus stimulates rapid epithelial restitution after injury by inducing Ca(2+) signaling.