423 Genome-Wide Epigenetic Determinants of Interstitial Cell of Cajal (ICC) Dysfunction
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421 Role of Wnt-β Catenin Signaling in Anal Sphincter Injury & Repair Mahadevan R. Rajasekaran, Valmik Bhargava, Mitra Salehi, Shantanu Sinha, Ravinder K. Mittal Background and Aims: Our recent studies to unravel pathophysiology of childbirth related injury to the anal sphincter muscle complex revealed anatomical disruptions1, increased fibrosis and impaired sphincter muscle function. Molecular mechanism of sphincter muscle fibrosis is unclear. Wnt-β catenin signaling pathway is recently recognized as the major molecular pathway involved in organ fibrosis2. The goal of our study was to specifically examine the role of this novel pathway in anal sphincter injury-related fibrosis and evaluate if intervention of this signaling pathway using a specific antagonist (SFRP2-secreted frizzled related protein-2) could attenuate fibrosis and improve EAS muscle function. Methods: New Zealand White female rabbits (n=12) were subjected to surgical myotomy by a cranio-caudal incision that extended along the entire length and thickness of the EAS muscle. Half of these animals received injection of Wnt antagonist (SFRP2- 2 μg/200μl, daily X 5 days) and the rest received saline (placebo group) at the myotomy site. Physiological measurements (anal canal pressure; length-tension function) were recorded at weekly intervals for 12 weeks post-myotomy. Animals were sacrificed at pre-determined time points and anal canal was harvested and processed for histochemical studies (Masson trichrome stain for muscle / connective tissue and Sirius red for collagen), immunostaining (β catenin) and molecular evaluation (Western blot & qPCR) for markers of fibrosis (collagen, connective tissue growth factor). Results: Surgical myotomy of the EAS resulted in significant impairment in anal canal pressure (Panel A-top) and L-T function. Immunostaining studies showed activation of Wnt-β catenin signaling pathway (Panel B) after injury. Molecular studies revealed 1.5 to 2 fold increase in levels of fibrosis markers (collagen, connective tissue growth factor). Treatment with SFRP-2 attenuated functional impairment (Panel A; as evidenced by improve- ment in anal canal pressure; Panel A-bottom), suppressed activation of β catenin (Panel B) and prevented fibrosis development (Panel C). Conclusions: Improvement in sphincter muscle function after selective inhibition of Wnt/β-catenin signaling suggests a novel thera- peutic potential for prevention of anal sphincter dysfunction consequent to sphincter injury. References: 1.Kim YS, Weinstein M, Raizada V, Jiang Y, Bhargava V, Rajasekaran MR, Mittal RK. Anatomical disruption and length-tension dysfunction of anal sphincter complex muscles in women with fecal incontinence. Dis Colon Rectum 2013;56:1282-9. 2.Akhmetshina A, Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, Horn A, Kireva T, Beyer C, Zwerina J, Schneider H, Sadowski A, Riener MO, MacDougald OA, Distler O, Schett G, Distler JH. Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. Nat Commun 2012;3:735. Effect of SFRP-2 on EAS Function 422 Effects of Mechanical Stress on Myenteric Neurons in the Colon You Min Lin, Feng Li, Barun Choudhury, John H. Winston, Sushil K. Sarna, Xuan-Zheng P. Shi Background and aim: Myenteric plexus (MP) between circular and longitudinal muscle layers plays a critical role in the control of gut motility. Previous studies suggested that neuronal nitric oxide synthase (nNOS) in MP is down-regulated, and the MP network is affected in obstructive bowel disorders (OBD). However, whether the alteration in MP network is a cause or consequence of OBD remains unknown. The present study tested the hypothesis that mechanical stress in obstruction disrupts the MP network and down-regulates nNOS gene expression. We also investigated the functional consequence of the alterations in MP. Methods: Mechanical stress was induced in vivo in a rat model of partial colon obstruction where a 21-mm obstruction band was implanted in the distal colon, 4 cm oral to the anus. This procedure increased lumen circumference of the oral segment from 20.5±0.4 to 31.1±0.5 mm (N = 5). In vitro mechanical stress was mimicked by static stretch of rat colonic muscle strips to 150% of their original lengths along the long axis of circular muscle. Results: (1) Immunofluorescence staining of the MP with PGP9.5 showed that the ENS network was disrupted profoundly in the distended colon on day 7 of obstruction. The number of nNOS positive enteric neurons was significantly reduced, compared to sham controls. (2) Western blot studies showed that PGP 9.5 protein level did not change in either the mechanically stressed segment oral to obstruction band, or in the non-distended aboral segment. However, nNOS expression was suppressed dramatically in a time-dependent manner in the distended oral segment. It started to decrease significantly on day 3 of obstruction. By day 7, nNOS level in the distended oral segment was only 29±10% of sham control (N = 5, p <0.01). The nNOS level did not show any significant change in the non-distended aboral segment. Real-time RT-PCR showed that nNOS mRNA level was also significantly decreased in the distended oral segment, but not in the non-stretched aboral segment. (3) In vitro stretch of colonic muscle strips for 24 hours did not alter PGP9.5 expression, but significantly suppressed nNOS expression to 65±13% of controls (N=6, p < 0.05). (4) Electrical field stimulation at 50 V and 1~ 5 Hz evoked frequency-dependent relaxation in colonic smooth muscle strips isolated from sham rats, which was blocked by NOS inhibitor L-NNA (100 μM). However, the EFS -evoked NOS mediated relaxation was almost completely abolished in the muscle strips isolated from the distended colon. The relaxation evoked by 2 Hz EFS S-91 AGA Abstracts was only 17±10.2% of the control (N = 4, p < 0.01). Conclusions: Mechanical stress disrupts colonic myenteric plexus network and down-regulates gene expression of nNOS in myenteric neurons. The down-regulated nNOS expression may account for impaired relaxation in pathological lumen distention. 423 Genome-Wide Epigenetic Determinants of Interstitial Cell of Cajal (ICC) Dysfunction Sabriya A. Syed, Jeong Heon Lee, Huihuang Yan, Zhiguo Zhang, Yujiro Hayashi, Tamas Ordog Background & Aims: ICC are reduced or their functions are dysregulated in several GI motor disorders. The mechanisms and reversibility of ICC dysfunction are unclear. We previously developed a model of dysfunctional ICC by creating clonal cell lines from murine gastric ICC (e.g., ICL2A) and maintaining them in the absence of Kit ligand. In these Kit low/ − cells lacking slow waves ("post-ICC") we found Kit transcription to be epigenetically repressed by trimethylation of histone 3 lysine 27 (H3K27me3). Experimental reduction of this histone mark restored not only Kit expression but also electrical slow waves suggesting that epigenetic repression may also underlie ICC dysfunction. Here, we aimed to identify the genes that are aberrantly repressed in post-ICC and the mechanisms responsible for their reduced transcription. Methods: Gene expression was studied in ICL2A post-ICC by Affymetrix Mouse Genome 430.2 microarrays and compared to data previously generated in freshly purified pacemaker and neuromodulator ICC (myenteric ICC; ICC-MY and deep muscular plexus ICC; ICC-DMP, respectively). Genome-wide distribution of activating (H3K4me3) and repressive histone marks (H3K27me3 and H3K9me3) was studied by chromatin immunoprecipitation-sequencing on Illumina HiSeq 2000. Results: 28% and 34% of the 542 and 403 genes uniquely expressed in ICC-DMP and ICC-MY, respectively, were downregulated in ICL2A cells. Compared to the entire genome, the average size of activating H3K4me3 peaks detected in the vicinity of the transcription start sites (TSS) of these genes was reduced ~3.5-fold, whereas the inhibitory H3K27me3 and H3K9me3 marks were increased 1.4- and 1.2-fold, respectively. Binding profiles of genes previously linked to ICC function indicated repression by H3K27me3 and H3K9me3 marks of the ion channel genes Ano1 and Cacna1h, which are critical for slow wave activity, as well as Chrm1,2,3 muscarinic receptors. In contrast, unopposed activating H3K4me3 peaks consistent with active transcription were found near the TSS of genes mediating nitrergic signaling (Prkg1,2, Gucy1a2) and several genes related to slow wave generation including genes involved in endoplasmic reticular Ca 2+ handling (Itpr1, Atp2a2), mitochondrial Ca 2+ handling (Mcu, Mcur1, Ccdc109b, Letm1, Slc8b1), regulation of transmembrane Ca 2+ flux including store- operated mechanisms (Orai1,2,3, Stim1,2, Cav1,2, Atp2b1), and the nonselective cation chan- nels Trpm7 and Ano6. Conclusions: Kit low/− post-ICC show transcriptional activation of several genes implicated in ICC function and their inability to generate rhythmic slow waves is likely due to the epigenetic repression of a subset of genes related to membrane ion transport. Inhibition of these repressive mechanisms may help restore ICC function in GI motor disorders. Support: NIH DK58185, Mayo Clinic Center for Individualized Medicine. 424 TLR9 Signaling Tunes Innate Immunity and Neuromuscular Function in Murine Small Intestine Paola Brun, Valentina Caputi, Lisa Spagnol, Giulia Schirato, Lucia Petrelli, Andrea Porzionato, Francesca Galeazzi, Giacomo C. Sturniolo, Ignazio Castagliuolo, Maria Cecilia Giron Background. Recently, it has been shown that signaling derived from microbial associated molecular patterns receptors such as Toll Like Receptor (TLR) 2 and 4 is required to maintain enteric nervous system (ENS) integrity and to prevent exaggerated inflammatory responses. Aim. This study was undertaken to assess whether signaling from TLR9 controls innate immunity and neuromuscular function in the small intestine. Methods. In male C57Bl/6 and TLR9 deficient (TLR9KO) mice (4-12 weeks old) we assessed: a) ileum integrity by light and electron microscopy (EM) examination; b) contractile activity of isolated ileum segments, vertically mounted in organ baths, as changes in isometric muscle tension following electric field stimulation (EFS, 1-40 Hz) in presence or absence of 1 μM tetrodotoxin or atropine; c) expression of neuronal HuC/D, and glial S100β proteins by double labelling confocal immunofluorescence in ileal whole mount preparations; d) ENS neurochemical code by neuronal nitric oxide synthase (nNOS) and substance P (SP) immunohistochemistry; e) mucosal macrophages, CD8- and CD4-lymhocytes activation by FACS analysis. Results. Light microscopy of ileal sections didn't evidenced gross anomalies in TLR9KO mice whereas EM analysis showed dilated cisternae of the endoplasmic reticulum (ER) in myenteric plexus neurons in absence of mitochondrial damage. Evidence of neuronal sufferance was supported by the severe reduction in HuC/D, a RNA-binding protein immunoreactivity and by the significant loss in nNOS+ and SP+ neurons and fibers in TLR9KO mice (p<0.01 vs control). However, S100β+ enteric glial cells were not affected in TLR9KO mice. Furthermore, enhanced neuronal cholinergic transmission (+45±6% at 20 Hz) was observed by in vitro contractility studies in TLR9KO mice. Activated mucosal macrophages (CD11c+F4/80+) but not CD4+IL4+ and CD8+IFNg+ cells were significantly increased (p<0.01) in TLR9KO mice. Conclusions. Our study provides evidence that in mice TLR9 signaling is required to tune enteric innate immune responses and to maintain ENS architecture, neurochemical coding and neuromuscular function. AGA Abstracts
Transcript of 423 Genome-Wide Epigenetic Determinants of Interstitial Cell of Cajal (ICC) Dysfunction
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Role of Wnt-β Catenin Signaling in Anal Sphincter Injury & RepairMahadevan R. Rajasekaran, Valmik Bhargava, Mitra Salehi, Shantanu Sinha, Ravinder K.Mittal
Background and Aims: Our recent studies to unravel pathophysiology of childbirth relatedinjury to the anal sphincter muscle complex revealed anatomical disruptions1, increasedfibrosis and impaired sphincter muscle function. Molecular mechanism of sphincter musclefibrosis is unclear. Wnt-β catenin signaling pathway is recently recognized as the majormolecular pathway involved in organ fibrosis2. The goal of our study was to specificallyexamine the role of this novel pathway in anal sphincter injury-related fibrosis and evaluateif intervention of this signaling pathway using a specific antagonist (SFRP2-secreted frizzledrelated protein-2) could attenuate fibrosis and improve EAS muscle function. Methods: NewZealand White female rabbits (n=12) were subjected to surgical myotomy by a cranio-caudalincision that extended along the entire length and thickness of the EAS muscle. Half ofthese animals received injection of Wnt antagonist (SFRP2- 2 μg/200μl, daily X 5 days) andthe rest received saline (placebo group) at the myotomy site. Physiological measurements(anal canal pressure; length-tension function) were recorded at weekly intervals for 12 weekspost-myotomy. Animals were sacrificed at pre-determined time points and anal canal washarvested and processed for histochemical studies (Masson trichrome stain for muscle /connective tissue and Sirius red for collagen), immunostaining (β catenin) and molecularevaluation (Western blot & qPCR) for markers of fibrosis (collagen, connective tissue growthfactor). Results: Surgical myotomy of the EAS resulted in significant impairment in analcanal pressure (Panel A-top) and L-T function. Immunostaining studies showed activationof Wnt-β catenin signaling pathway (Panel B) after injury. Molecular studies revealed 1.5to 2 fold increase in levels of fibrosis markers (collagen, connective tissue growth factor).Treatment with SFRP-2 attenuated functional impairment (Panel A; as evidenced by improve-ment in anal canal pressure; Panel A-bottom), suppressed activation of β catenin (Panel B)and prevented fibrosis development (Panel C). Conclusions: Improvement in sphinctermuscle function after selective inhibition of Wnt/β-catenin signaling suggests a novel thera-peutic potential for prevention of anal sphincter dysfunction consequent to sphincter injury.References: 1.Kim YS, Weinstein M, Raizada V, Jiang Y, Bhargava V, Rajasekaran MR, MittalRK. Anatomical disruption and length-tension dysfunction of anal sphincter complex musclesin women with fecal incontinence. Dis Colon Rectum 2013;56:1282-9. 2.Akhmetshina A,Palumbo K, Dees C, Bergmann C, Venalis P, Zerr P, Horn A, Kireva T, Beyer C, ZwerinaJ, Schneider H, Sadowski A, Riener MO, MacDougald OA, Distler O, Schett G, Distler JH.Activation of canonical Wnt signalling is required for TGF-beta-mediated fibrosis. NatCommun 2012;3:735.
Effect of SFRP-2 on EAS Function
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Effects of Mechanical Stress on Myenteric Neurons in the ColonYou Min Lin, Feng Li, Barun Choudhury, John H. Winston, Sushil K. Sarna, Xuan-ZhengP. Shi
Background and aim: Myenteric plexus (MP) between circular and longitudinal muscle layersplays a critical role in the control of gut motility. Previous studies suggested that neuronalnitric oxide synthase (nNOS) in MP is down-regulated, and the MP network is affected inobstructive bowel disorders (OBD). However, whether the alteration in MP network is acause or consequence of OBD remains unknown. The present study tested the hypothesisthat mechanical stress in obstruction disrupts the MP network and down-regulates nNOSgene expression. We also investigated the functional consequence of the alterations in MP.Methods: Mechanical stress was induced in vivo in a rat model of partial colon obstructionwhere a 21-mm obstruction band was implanted in the distal colon, 4 cm oral to the anus.This procedure increased lumen circumference of the oral segment from 20.5±0.4 to 31.1±0.5mm (N = 5). In vitro mechanical stress was mimicked by static stretch of rat colonic musclestrips to 150% of their original lengths along the long axis of circular muscle. Results: (1)Immunofluorescence staining of the MP with PGP9.5 showed that the ENS network wasdisrupted profoundly in the distended colon on day 7 of obstruction. The number of nNOSpositive enteric neurons was significantly reduced, compared to sham controls. (2) Westernblot studies showed that PGP 9.5 protein level did not change in either the mechanicallystressed segment oral to obstruction band, or in the non-distended aboral segment. However,nNOS expression was suppressed dramatically in a time-dependent manner in the distendedoral segment. It started to decrease significantly on day 3 of obstruction. By day 7, nNOSlevel in the distended oral segment was only 29±10% of sham control (N = 5, p <0.01).The nNOS level did not show any significant change in the non-distended aboral segment.Real-time RT-PCR showed that nNOS mRNA level was also significantly decreased in thedistended oral segment, but not in the non-stretched aboral segment. (3) In vitro stretchof colonic muscle strips for 24 hours did not alter PGP9.5 expression, but significantlysuppressed nNOS expression to 65±13% of controls (N=6, p < 0.05). (4) Electrical fieldstimulation at 50 V and 1~ 5 Hz evoked frequency-dependent relaxation in colonic smoothmuscle strips isolated from sham rats, which was blocked by NOS inhibitor L-NNA (100μM). However, the EFS -evoked NOS mediated relaxation was almost completely abolishedin the muscle strips isolated from the distended colon. The relaxation evoked by 2 Hz EFS
S-91 AGA Abstracts
was only 17±10.2% of the control (N = 4, p < 0.01). Conclusions: Mechanical stress disruptscolonic myenteric plexus network and down-regulates gene expression of nNOS in myentericneurons. The down-regulated nNOS expression may account for impaired relaxation inpathological lumen distention.
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Genome-Wide Epigenetic Determinants of Interstitial Cell of Cajal (ICC)DysfunctionSabriya A. Syed, Jeong Heon Lee, Huihuang Yan, Zhiguo Zhang, Yujiro Hayashi, TamasOrdog
Background & Aims: ICC are reduced or their functions are dysregulated in several GImotor disorders. The mechanisms and reversibility of ICC dysfunction are unclear. Wepreviously developed a model of dysfunctional ICC by creating clonal cell lines from murinegastric ICC (e.g., ICL2A) and maintaining them in the absence of Kit ligand. In these Kitlow/
− cells lacking slow waves ("post-ICC") we found Kit transcription to be epigeneticallyrepressed by trimethylation of histone 3 lysine 27 (H3K27me3). Experimental reduction ofthis histone mark restored not only Kit expression but also electrical slow waves suggestingthat epigenetic repression may also underlie ICC dysfunction. Here, we aimed to identifythe genes that are aberrantly repressed in post-ICC and the mechanisms responsible fortheir reduced transcription. Methods: Gene expression was studied in ICL2A post-ICC byAffymetrix Mouse Genome 430.2 microarrays and compared to data previously generatedin freshly purified pacemaker and neuromodulator ICC (myenteric ICC; ICC-MY and deepmuscular plexus ICC; ICC-DMP, respectively). Genome-wide distribution of activating(H3K4me3) and repressive histone marks (H3K27me3 and H3K9me3) was studied bychromatin immunoprecipitation-sequencing on Illumina HiSeq 2000. Results: 28% and34% of the 542 and 403 genes uniquely expressed in ICC-DMP and ICC-MY, respectively,were downregulated in ICL2A cells. Compared to the entire genome, the average size ofactivating H3K4me3 peaks detected in the vicinity of the transcription start sites (TSS) ofthese genes was reduced ~3.5-fold, whereas the inhibitory H3K27me3 and H3K9me3 markswere increased 1.4- and 1.2-fold, respectively. Binding profiles of genes previously linkedto ICC function indicated repression by H3K27me3 and H3K9me3 marks of the ion channelgenes Ano1 and Cacna1h, which are critical for slow wave activity, as well as Chrm1,2,3muscarinic receptors. In contrast, unopposed activating H3K4me3 peaks consistent withactive transcription were found near the TSS of genes mediating nitrergic signaling (Prkg1,2,Gucy1a2) and several genes related to slow wave generation including genes involved inendoplasmic reticular Ca2+ handling (Itpr1, Atp2a2), mitochondrial Ca2+ handling (Mcu,Mcur1, Ccdc109b, Letm1, Slc8b1), regulation of transmembrane Ca2+ flux including store-operated mechanisms (Orai1,2,3, Stim1,2, Cav1,2, Atp2b1), and the nonselective cation chan-nels Trpm7 and Ano6. Conclusions: Kitlow/− post-ICC show transcriptional activation ofseveral genes implicated in ICC function and their inability to generate rhythmic slow wavesis likely due to the epigenetic repression of a subset of genes related to membrane iontransport. Inhibition of these repressive mechanisms may help restore ICC function in GImotor disorders. Support: NIH DK58185, Mayo Clinic Center for Individualized Medicine.
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TLR9 Signaling Tunes Innate Immunity and Neuromuscular Function inMurine Small IntestinePaola Brun, Valentina Caputi, Lisa Spagnol, Giulia Schirato, Lucia Petrelli, AndreaPorzionato, Francesca Galeazzi, Giacomo C. Sturniolo, Ignazio Castagliuolo, Maria CeciliaGiron
Background. Recently, it has been shown that signaling derived from microbial associatedmolecular patterns receptors such as Toll Like Receptor (TLR) 2 and 4 is required to maintainenteric nervous system (ENS) integrity and to prevent exaggerated inflammatory responses.Aim. This study was undertaken to assess whether signaling from TLR9 controls innateimmunity and neuromuscular function in the small intestine. Methods. In male C57Bl/6and TLR9 deficient (TLR9KO) mice (4-12 weeks old) we assessed: a) ileum integrity bylight and electron microscopy (EM) examination; b) contractile activity of isolated ileumsegments, vertically mounted in organ baths, as changes in isometric muscle tension followingelectric field stimulation (EFS, 1-40 Hz) in presence or absence of 1 μM tetrodotoxin oratropine; c) expression of neuronal HuC/D, and glial S100β proteins by double labellingconfocal immunofluorescence in ileal whole mount preparations; d) ENS neurochemicalcode by neuronal nitric oxide synthase (nNOS) and substance P (SP) immunohistochemistry;e) mucosal macrophages, CD8- and CD4-lymhocytes activation by FACS analysis. Results.Light microscopy of ileal sections didn't evidenced gross anomalies in TLR9KO mice whereasEM analysis showed dilated cisternae of the endoplasmic reticulum (ER) in myenteric plexusneurons in absence of mitochondrial damage. Evidence of neuronal sufferance was supportedby the severe reduction in HuC/D, a RNA-binding protein immunoreactivity and by thesignificant loss in nNOS+ and SP+ neurons and fibers in TLR9KO mice (p<0.01 vs control).However, S100β+ enteric glial cells were not affected in TLR9KO mice. Furthermore,enhanced neuronal cholinergic transmission (+45±6% at 20 Hz) was observed by in vitrocontractility studies in TLR9KO mice. Activated mucosal macrophages (CD11c+F4/80+) butnot CD4+IL4+ and CD8+IFNg+ cells were significantly increased (p<0.01) in TLR9KO mice.Conclusions. Our study provides evidence that in mice TLR9 signaling is required to tuneenteric innate immune responses and to maintain ENS architecture, neurochemical codingand neuromuscular function.
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