- Email: [email protected]
Sacral Nerve Stimulation Alters the Frequency of Colon Propagating Sequences in Patients With Neurogenic Fecal Incontinence
Table 1: Rectal ST (sensory threshold) and PT (pain threshold) in mA following 1 Hz, 10 Hz and sham rLSMS, for baseline and 0, 30 and 60 minutes post stimulation. Table 2
997 Comparison of Angiogenic Growth Factors to Optimize Physiological Function of Implanted Tissue Engineered Internal Anal Sphincter (IAS) Shreya Raghavan, Eiichi A. Miyasaka, Robert R. Gilmont, Daniel H. Teitelbaum, Khalil N. Bitar
Table 2: Anal sphincter MEP in microvolts following 1 Hz, 10 Hz and sham rLSMS, for baseline and 30 minutes post stimulation.
Background: We have previously successfully implanted bioengineered IAS tissues in a mouse model. Neovascularization is key to the survival of implanted bioengineered constructs. We previously found no significant difference in neovascularization, In Vivo growth, implanted muscle tissue diameter and viability with any of the three growth factors - fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). Objective: We aim to evaluate the physiology of the implanted bioengineered IAS when supplied with these different angiogenic growth factors, in order to: i) optimize the maintenance of physiological characteristics along with phenotype; ii) evaluate the efficacy of the FDA-approved growth factor, PDGF, in physiological function of IAS smooth muscle. Methods: Bioengineered IAS tissue constructs were implanted on the dorsum of C57BL/6 mice for 25 days. A micro-osmotic pump primed with FGF, VEGF or PDGF-BB was implanted to locally supply growth factor to the implanted tissue. Reported values are means ± SEM. Results: Histology of harvested post-implant IAS revealed neovascularization with all three growth factors. The number of blood vessels per high power field was not significantly different between the three growth factors. (1) Myogenic spontaneous basal tone was generated in all three groups of tissues; (2) Nifedipine blocked generation of myogenic basal tone by up to 80% with all three growth factors; (3) Vasoactive Intestinal Peptide(VIP) caused a relaxation of basal tone up to 68 ± 25 % of basal tone (PDGF), 77 ± 5.1 % (VEGF), compared to 84 ± 9% (FGF). (4) Acetylcholine(Ach) response was dose-dependent and elicited rapid-rising, sustained contractions of up to 200μN (PDGF), 275μN (VEGF) and 150μN (FGF); (5) KCl treatment generated rapid rising contractions, quantified as a percentage of agonist (Ach)-induced contraction. Implanted IAS generated up to 93.82 ± 24% (PDGF), 82.51 ± 18% (VEGF) and 96.27 ± 2.4% (FGF) of Ach-induced contraction. Summary: Bioengineered IAS tissue constructs displayed all key aspects of IAS physiology postimplantation. The growth factor used (VEGF, PDGF or FGF) did not significantly alter the physiological characteristics, allowing for the use of an FDA-approved substance in the implantation of tissue engineered constructs. Conclusion: The use of angiogenic growth factors (VEGF, PDGF or FGF) all resulted in neovascularization of implanted tissue engineered IAS. PDGF-BB, an FDA-approved substance, has shown remarkable efficacy in the maintenance of the IAS smooth muscle In Vivo. This confirms that the use of autologous bioengineered IAS is a viable route for fecal incontinence therapeutics. Supported by NIHRO1DK071614 and 1RC1DK087151.
999 Is the Accuracy of Anorectal Manometry (ARM) Worthwile in Patients With Fecal Incontinence (FI) Sabrina Labermeyer, Holger Seidl, Nicola Scalercio, Felix Gundling, Thomas Schmidt, Wolfgang Schepp, Christian Pehl Background and Aim: FI patients have lower mean anal resting (MRP) and squeeze (MSP) pressure and an impaired sensitivity compared to healthy people. However, to be useful in clinical routine, a test must separate precisely between health and disease. Some investigators state that differentiation between FI and continent patients was not possible with ARM because there was complete overlap of the data ranges (Dis Colon Rectum 1990;33:47985), while others found that 92% of patients had a pathological MSP (Gut 1992;33:80713). The aim of the present study was to evaluate the accuracy of ARM in a huge cohort of FI patients and controls. Material and Methods: Normal ARM data were obtained from 144 healthy people (71 women; median age 63(21-90)yrs) and compared with 573 FI patients (416 women; 63(19-94)yrs). FI for gas (grade I) was seen in 153, for liquids (grade II) in 272, and for solid stool (grade III) in 213 patients. Mean resting (MRP) and squeeze (MSP) pressure, and balloon volume at first perception (BVP) and at urge sensation (BVU) were determined. A score was calculated from the ARM data by logistic regression analysis taking into account the discriminatory power of each ARM parameter (see appendix). ROC analysis was used to determine optimal cut offs between healthy people and FI patients. Sensitivity, specificity and accuracy (true positives + true negatives / patients + probands) was calculated for each parameter as well as for ARM (at least one pathological parameter or a pathological score). Results:The best sensitivity and accuracy was determined for the score without significantly decreasing the specifity (tab.1). The pressure data showed higher sensitivity and accuracy than the sensory data despite comparable specifity. ARM demonstrated an excellent sensitivity and accuracy, but had only a moderate specificity (tab.2). The sensitivity of ARM increased with increasing severity of FI. Conclusion: Sensitivity and accuracy of the single ARM parameter is only moderate for the pressure data and poor for the sensory data. The best accuracy showed a weighted score which should be further evaluated prospectively in a second cohort. In contrast to the single parameter, ARM demonstrated an excellent sensitivity, a moderate specifity, and a convincing accuracy. appendix: f(x) = exp(4,429-0,012*MRP-0,013*MSP+0,03*BVP-0,014*MSP)/1+exp(4,4290,012*MRP-0,013*MSP+0,03*BVP-0,014*MSP) accuracy of ARM parameter
998 Sensorimotor Modulation of the Human Brain-Gut Axis Induced by NonInvasive Lumbosacral Magnetic Stimulation Tarig Algladi, Mary Louise Harris, Peter J. Whorwell, Shaheen Hamdy Background: Functional gut and defecation disorders are common yet difficult to manage and brain-gut axis dysfunction has been implicated in both. A better understanding of braingut physiology is of importance in designing new treatments to improve function. One such method is non-invasive magnetic stimulation (MS) which can modulate the nervous system and may have a role in restoring brain-gut function. The aim of this study is to ascertain whether MS applied to the lumbosacrum can alter human brain gut function, using rectal sensitivity and cortico-anal excitability as our model. Methods: Participants: 13 healthy volunteers (7 females, age range 20-56 years) participated in this study. Sensory measurements were performed via an electrode stimulating catheter positioned in the rectum 10 cm from the anal verge. Sensory and pain thresholds were determined using electrical stimuli [3]. The assessments of sensory and pain thresholds of the rectum were performed at baseline and then immediately, 30 and 60 min after each intervention. Anal sphincter EMG responses were recorded from an anal plug following single pulse transcranial magnetic stimulation over the motor cortex (TMS) and lumbosacrum (LSMS), at baseline and 30 min following each intervention. Interventions comprised 3 different neurostimulation regimens delivered in random order over separate days as repetitive (1 Hz, 10 Hz and sham) lumbosacral magnetic stimulation (rLSMS). Data were (mean±SEM) analysed by two-way ANOVA. Results: Compared to sham, there was a significant increase in sensory and pain thresholds in the rectum in the hour after both 1 Hz rLSMS and 10 Hz rLSMS (*p<0.01, Table 1) By contrast, only 1 Hz rLSMS was accompanied by a significant increase in lumbosacral motor excitability of the anal sphincter (**p<0.05, Table 2). Conclusion: The application of magnetic stimulation to lumbosacral area is able to modulate human visceral sensitivity and corticoanal motor excitability in a frequency dependent manner and holds the promise of being applied to patients with functional GI and defecation disorders as a future therapeutic intervention. Table 1
accuracy of anorectal manometry
1000 Sacral Nerve Stimulation Alters the Frequency of Colon Propagating Sequences in Patients With Neurogenic Fecal Incontinence Vicki E. Patton, John W. Arkwright, David Z. Lubowski, Philip G. Dinning Background: Fecal incontinence (FI) is a chronic condition causing major economic and social burdens. Sacral nerve stimulation (SNS) has emerged as an effective treatment for patients with this condition. However the mode of action by which SNS improves continence remains unclear. Aims: To examine the impact of SNS on colonic propagating sequences (PS) in patients with neurogenic fecal incontinence using our newly developed fibre-optic high resolution manometry catheter in a pilot study in four patients. Methods: Under general
S-161
AGA Abstracts
AGA Abstracts
contraction up to 90μN; (ii) Relaxation of up to 50μN was induced either by Vasoactive Intestinal Peptide (VIP) or 8-bromo-cyclicAMP. Summary: Circular smooth muscle cells maintained viability when cultured on chitosan-coated tissue culture plates and preserved their normal spindle-like shape and expression of contractile smooth muscle markers. Bioengineered tissue constructs harvested from the scaffold maintained their contraction and relaxation responses compared to control tissue constructs that were not placed on the scaffold. Conclusion: Chitosan is non-toxic to colonic smooth muscle cells and provided a good matrix for their survival and maintenance of their contractile phenotype. The tissue constructs maintained the integrity of receptors for VIP and Acetylcholine, and the integrity of intracellular signaling pathways for contraction and relaxation. This is the first report of bioengineering colonic circular smooth muscle tissue constructs on biodegradable chitosan scaffolds. This study provides a basis for creating a functional colon with the possibility of building a longitudinal smooth muscle layer on top of the bioengineered circular layer. Supported by NIH1RC1DK087151.
AGA Abstracts
anesthesia, a fibre-optic manometry catheter (90 recording sites @ 1cm intervals) was positioned colonoscopically, with the tip clipped to the caecum to prevent displacement. A four-electrode tined lead was implanted in 3 patients and a single electrode (Medtronic, Sydney) in one patient, into the S3 sacral nerve foramin and correct placement confirmed by intra-operative xray and nerve stimulation eliciting pelvic floor contraction. After a 2hr basal recording in the fasted state, in a single-blind randomized fashion patients had either sham stimulation or supra-sensory stimulation (14Hz; 300msec). Data are expressed as mean deltas (i.e. stimulation frequency - basal frequency) Results: First we examined the change in frequency of antegrade and retrograde PSs throughout the entire colon during the sham and SNS stimulation. In comparison to sham stimulation, SNS increased the frequency of both antegrade (sham; -2.0 ± 5.5 vs SNS; 9.7 ± 16 PS/2hr) and retrograde (sham; 3.3 ± 6.8 vs SNS; 18 ± 9.1 PS/2hr) propagating sequences. We then looked specifically for changes in the frequency of PSs in the left colon (descending colon and sigmoid), and in comparison to sham stimulation SNS resulted in an increase in the frequency of retrograde PSs (sham; -3 ± 5.6 vs SNS 23 ± 3.6 PS/hr). Conclusion: Sacral nerve stimulation increases both antegrade and retrograde propagating sequences in patients with fecal incontinence. Of potential interest is the increase in retrograde PSs in the distal colon. These data suggest that one mode of action for SNS improving continence is through the alteration of colonic motility.
1003 IGFBP3 Suppresses Reactive Oxygen Species and Cellular Senescence Response to Facilitate TGF-β-Mediated EMT Mitsuteru Natsuizaka, Shinya Ohashi, Azal Ahmadi, Sanders Chang, Kei Nakagawa, Gabrielle S. Wong, Nobuki Miyamoto, Katharine D. Grugan, Andres J. Klein-Szanto, Meenhard Herlyn, J. Alan Diehl, Hiroshi Nakagawa Introduction: Insulin-like growth factor (IGF) binding protein-3 (IGFBP3), a transcriptional target for hypoxia inducible factor (HIF)-1α, is frequently overexpressed in esophageal carcinomas. IGFBP3 facilitates TGF-β-mediated epithelial-to-mesenchymal transition (EMT) in an IGF-independent manner in transformed human esophageal cells (Carcinogenesis. 2010;31:1344-53). Our work indicates that cellular senescence checkpoints need to be suppressed during EMT (Cancer Res. 2010;70:4174-84). We hypothesized that IGFBP3 may alleviate reactive-oxygen species (ROS)-mediated cellular stresses. Methods: Esophageal cancer cell lines and EMT-competent human esophageal epithelial cells transformed by combinations of epidermal growth factor receptor (EGFR) and p53R175H were stably transduced with wild-type (WT) or I56G/L80G/L81G (GGG) mutant IGFBP3, the latter incapable of binding IGFs. Short hairpin RNAs (shRNAs) were used to knockdown IGFBP3. Gene expression was determined by real-time RT-PCR and Western blotting. Senescence-associated β-galactosidase activity was determined. Intracellular ROS level was measured with 2′-7′dichlorodihydrofluorescein diacetate as a fluorescent probe in flow cytometry. Results: Hypoxia (1% O2) induced concomitantly HIF-1α and IGFBP3 in all esophageal cancer cell lines tested (n=10) without eliciting growth inhibitory effects. Interestingly, either hypoxia or exogenous H2O2 induced ROS to a lesser extent in cells expressing a higher level of IGFBP3, implying IGFBP3 in negative regulation of ROS. Upon exposure to hypoxia or H2O2, IGFBP3 knockdown augmented ROS while ectopic expression of either WT or GGGIGFBP3 reduced ROS, indicating an IGF-independent IGFBP3 effect upon ROS generation. Unlike hypoxia, H2O2 reduced cancer cell viability in a dose-dependent manner, and that was augmented by IGFBP3 knockdown and antagonized by ectopic IGFPB3 expression. In EMT-competent transformed cells, TGF-β induced IGFBP3 and EMT, as corroborated by spindle-shaped cell morphology (>80%) and an E-cadherin to N-cadherin class switch, but senescence to a limited extent (<10%). By contrast, TGF-β induced a higher level of ROS and senescence, but not IGFBP3, in empty vector transduced non-transformed control cells that appeared to be less EMT-competent (<20%), where Trolox, a pharmacological antioxidant reduced senescence implying TGF-β-induction of ROS. Conclusions: These mechanistic data suggest that IGFBP3 may have a novel IGF-independent role in the hypoxic tumor microenvironment to alleviate ROS-mediated cellular stresses and senescence to modulate cellular plasticity. This has implications upon EMT in other cancers and may offer platforms for cancer therapy.
1001 CD95 Signaling Induces EMT in Colon Cancer Cells Hanchen Li, Haoxuan Zheng, Jian Hua Liu, Jean Marie Houghton Background The CD95/Fas Ag apoptosis signaling pathway can be used by various cell types for non- apoptotic signaling. Our group has shown that colon cancer cells acquire CD95 apoptosis resistance and gain CD95L expression as they progress from locally invasive disease to metastatic disease. Here we investigate the role of CD95 in the epithelial to mesenchymal transition as cancer cells become invasive. Methods The MC38 colon cancer cell line and the derivatives MC38/FLIP and MC38/FLIP/L have been previously published. The MC38 cell line expresses CD95 on the cell surface and is susceptible to ligand induced apoptosis. MC38/FLIP express the anti-apoptotic protein-FLIP and are resistant to ligand induced apoptosis, but instead proliferate in response to addition of exogenous ligand. MC38/FLIP/ L cells are also resistant to apoptosis however they constitutively express ligand and are more aggressive and proliferate at a higher rate under baseline conditions without the need of additional ligand. We evaluated these cells with and without FasL for the expression of mesenchymal and epithelial specific markers and for EMT specific transcription factors by RT-PCR and Western blot analysis. Results. MC38/FLIP cells assume a more spindle shape in culture when grown in the presence of ligand. Vimentin expression is markedly increased with the addition of ligand. Interestingly, E-cadherin is decreased and N-cadherin expression is markedly increased while B-catenin undergoes relocation to the nucleus with activation of the CD95 pathway in apoptosis resistant colon cancer cells. Conclusion Our findings suggest that in addition to increasing proliferation, CD95 signaling in colon cancer may underlie the EMT important for invasion and metastasis. These findings warn that therapies aimed at inducing apoptosis through classical pathways such as CD95 may need to be approached cautiously, as this pathway may be responsible the aggressive properties of tumors.
1004 Epithelial-to-Mesenchymal Transition and Hematogenous Dissemination Precedes Histologic Diagnosis of Cancer in a Genetic Mouse Model of Pancreatic Ductal Adenocarcinoma Andrew D. Rhim, Ben Stanger BACKGROUND: Less than 20% of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) present with primary tumors of limited size and no clinical evidence of metastasis. Most of these patients undergo pancreatic resection. However, almost all of these patients eventually die of metastatic disease to distant organs. These phenomena suggest that PDAC cell dissemination may precede tumor formation. Here, using a unique and reliable lineage-labeled mouse model of spontaneous PDAC, we sought to determine if epithelial-to-mesenchymal transition (EMT) and hematogenous dissemination occurred at the precancerous pancreatic intraepithelial neoplasm (PanIN) stage. METHODS: We bred Pdx-Cre; LSL-KrasG12D; p53fl/+ or Ink4afl/+; Rosa26LSL-YFP mice in which only pancreatic epithelial cells are permanently labeled with yellow fluorescent protein (YFP). Pancreas and blood specimens from mice aged 2-2.5 mo (mice with only the precancerous PanIN lesion and no histologic evidence of cancer by H&E) and 3.5-4 mo (all with tumors, PDAC) were analyzed. RESULTS: >95% of all pancreatic epithelial cells were labeled with YFP in control Pdx-Cre;RosaYFP mice. Multicolor immunofluorecent (IF) staining for the YFP epithelial lineage label and the mesenchymal markers Zeb1 and FSP1 revealed double positive (EMT+) cells residing in 2.2% and 17.4% of all PanIN2 and PanIN3 lesions, respectively, but in no control Pdx-Cre; RosaYFP mice. Individual spindle-shaped YFP+ cells were observed invading into the pancreatic stroma of PanIN mice, despite the absence of a pathologic diagnosis of cancer based on H&E staining. Using FACS, YFP+ pancreatic cells were found in the blood of both tumor-bearing PDAC mice and PanIN mice (94.5 ± 35.4 v. 42.9 ± 20.5 cells/ml blood; p<0.001; n=30) but not in control Pdx-Cre;RosaYFP mice. These circulating YFP+ cells were devoid of CD45 and Ter-119 (markers of white and red blood cells); expressed YFP, E-cadherin and Pdx-1 transcripts; and formed pancreatospheres in attachment-free cultures. Finally, while rare individual YFP+ cells were found in small hepatic venules of PanIN mice, no metastatic lesions were appreciated, as seen in PDAC mice. CONCLUSIONS: With the use of lineage labeling technology, we provide evidence that EMT, invasion, and hematogenous dissemination may precede histologic diagnosis of PDAC. These data argue against the classical linear progression model of cancer, whereby dissemination occurs after tumors form. However, it is unclear if circulating PanIN cells are the source of metastatic lesions. Nevertheless, based on our data, circulating pancreatic cells may be specific biomarkers for advanced PanIN disease in humans. Further studies to confirm if these data translate to humans are underway.
1002 MTORC1 and mTORC2 Regulate EMT, Motility and Metastasis of Colorectal Cancer via RHOA and RAC1 Signaling Pathways Pat Gulhati, Kanika A. Bowen, Jianyu Liu, Payton D. Stevens, Piotr Rychahou, Min Chen, Eun Y. Lee, Heidi Weiss, Kathleen L. O'Connor, Tianyan Gao, B. M. Evers Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is associated with growth and progression of colorectal cancer (CRC). The mammalian target of rapamycin (mTOR) kinase acts downstream of PI3K and nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translational initiation through the effectors S6K and 4E-BP1. The mTOR-RICTOR complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin allosterically inhibits the kinase activity of mTORC1, while mTORC2 is believed to be rapamycininsensitive. The contribution of mTOR and its interaction partners in regulating CRC progression and metastasis remains poorly understood. The purpose of the present study was to determine the role of mTORC1 and mTORC2 in regulating EMT, motility and metastasis of CRC. METHODS. HCT116, KM20 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with shRNA directed against RAPTOR or RICTOR. Effects on migration and invasion were analyzed using transwell and wound healing assays. Changes in actin cytoskeleton and cell-cell contacts were determined by immunofluorescence microscopy. RhoA and Rac1 activation was determined using GST-TRBD and GST-PBD pulldown assays, respectively. Establishment of metastasis was determined using intravenous injection of CRC cells. RESULTS. (i) We found that increased expression of mTOR, Raptor and Rictor mRNA was noted with more advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastasis compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116, KM20 and SW480 CRC cells. (iii) Stable shRNA-mediated knockdown of Raptor and Rictor significantly decreased the migration and invasion of HCT116, KM20 and SW480 cells. (iv) Stable shRNA-mediated knockdown of Raptor and Rictor leads to mesenchymal-to-epithelial transition and enhances sensitivity to oxaliplatin-induced apoptosis. (v) Stable shRNA-mediated knockdown of Raptor and Rictor leads to characteristic rearrangements in actin cytoskeleton concomitant with decreased RhoA and Rac1 activation. (vi) Establishment of CRC metastasis In Vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. CONCLUSIONS. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating migration, invasion, EMT and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC.
AGA Abstracts
S-162
997 Comparison of Angiogenic Growth Factors to Optimize Physiological Function of Implanted Tissue Engineered Internal Anal Sphincter (IAS) Shreya Raghavan, Eiichi A. Miyasaka, Robert R. Gilmont, Daniel H. Teitelbaum, Khalil N. Bitar
Table 2: Anal sphincter MEP in microvolts following 1 Hz, 10 Hz and sham rLSMS, for baseline and 30 minutes post stimulation.
Background: We have previously successfully implanted bioengineered IAS tissues in a mouse model. Neovascularization is key to the survival of implanted bioengineered constructs. We previously found no significant difference in neovascularization, In Vivo growth, implanted muscle tissue diameter and viability with any of the three growth factors - fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). Objective: We aim to evaluate the physiology of the implanted bioengineered IAS when supplied with these different angiogenic growth factors, in order to: i) optimize the maintenance of physiological characteristics along with phenotype; ii) evaluate the efficacy of the FDA-approved growth factor, PDGF, in physiological function of IAS smooth muscle. Methods: Bioengineered IAS tissue constructs were implanted on the dorsum of C57BL/6 mice for 25 days. A micro-osmotic pump primed with FGF, VEGF or PDGF-BB was implanted to locally supply growth factor to the implanted tissue. Reported values are means ± SEM. Results: Histology of harvested post-implant IAS revealed neovascularization with all three growth factors. The number of blood vessels per high power field was not significantly different between the three growth factors. (1) Myogenic spontaneous basal tone was generated in all three groups of tissues; (2) Nifedipine blocked generation of myogenic basal tone by up to 80% with all three growth factors; (3) Vasoactive Intestinal Peptide(VIP) caused a relaxation of basal tone up to 68 ± 25 % of basal tone (PDGF), 77 ± 5.1 % (VEGF), compared to 84 ± 9% (FGF). (4) Acetylcholine(Ach) response was dose-dependent and elicited rapid-rising, sustained contractions of up to 200μN (PDGF), 275μN (VEGF) and 150μN (FGF); (5) KCl treatment generated rapid rising contractions, quantified as a percentage of agonist (Ach)-induced contraction. Implanted IAS generated up to 93.82 ± 24% (PDGF), 82.51 ± 18% (VEGF) and 96.27 ± 2.4% (FGF) of Ach-induced contraction. Summary: Bioengineered IAS tissue constructs displayed all key aspects of IAS physiology postimplantation. The growth factor used (VEGF, PDGF or FGF) did not significantly alter the physiological characteristics, allowing for the use of an FDA-approved substance in the implantation of tissue engineered constructs. Conclusion: The use of angiogenic growth factors (VEGF, PDGF or FGF) all resulted in neovascularization of implanted tissue engineered IAS. PDGF-BB, an FDA-approved substance, has shown remarkable efficacy in the maintenance of the IAS smooth muscle In Vivo. This confirms that the use of autologous bioengineered IAS is a viable route for fecal incontinence therapeutics. Supported by NIHRO1DK071614 and 1RC1DK087151.
999 Is the Accuracy of Anorectal Manometry (ARM) Worthwile in Patients With Fecal Incontinence (FI) Sabrina Labermeyer, Holger Seidl, Nicola Scalercio, Felix Gundling, Thomas Schmidt, Wolfgang Schepp, Christian Pehl Background and Aim: FI patients have lower mean anal resting (MRP) and squeeze (MSP) pressure and an impaired sensitivity compared to healthy people. However, to be useful in clinical routine, a test must separate precisely between health and disease. Some investigators state that differentiation between FI and continent patients was not possible with ARM because there was complete overlap of the data ranges (Dis Colon Rectum 1990;33:47985), while others found that 92% of patients had a pathological MSP (Gut 1992;33:80713). The aim of the present study was to evaluate the accuracy of ARM in a huge cohort of FI patients and controls. Material and Methods: Normal ARM data were obtained from 144 healthy people (71 women; median age 63(21-90)yrs) and compared with 573 FI patients (416 women; 63(19-94)yrs). FI for gas (grade I) was seen in 153, for liquids (grade II) in 272, and for solid stool (grade III) in 213 patients. Mean resting (MRP) and squeeze (MSP) pressure, and balloon volume at first perception (BVP) and at urge sensation (BVU) were determined. A score was calculated from the ARM data by logistic regression analysis taking into account the discriminatory power of each ARM parameter (see appendix). ROC analysis was used to determine optimal cut offs between healthy people and FI patients. Sensitivity, specificity and accuracy (true positives + true negatives / patients + probands) was calculated for each parameter as well as for ARM (at least one pathological parameter or a pathological score). Results:The best sensitivity and accuracy was determined for the score without significantly decreasing the specifity (tab.1). The pressure data showed higher sensitivity and accuracy than the sensory data despite comparable specifity. ARM demonstrated an excellent sensitivity and accuracy, but had only a moderate specificity (tab.2). The sensitivity of ARM increased with increasing severity of FI. Conclusion: Sensitivity and accuracy of the single ARM parameter is only moderate for the pressure data and poor for the sensory data. The best accuracy showed a weighted score which should be further evaluated prospectively in a second cohort. In contrast to the single parameter, ARM demonstrated an excellent sensitivity, a moderate specifity, and a convincing accuracy. appendix: f(x) = exp(4,429-0,012*MRP-0,013*MSP+0,03*BVP-0,014*MSP)/1+exp(4,4290,012*MRP-0,013*MSP+0,03*BVP-0,014*MSP) accuracy of ARM parameter
998 Sensorimotor Modulation of the Human Brain-Gut Axis Induced by NonInvasive Lumbosacral Magnetic Stimulation Tarig Algladi, Mary Louise Harris, Peter J. Whorwell, Shaheen Hamdy Background: Functional gut and defecation disorders are common yet difficult to manage and brain-gut axis dysfunction has been implicated in both. A better understanding of braingut physiology is of importance in designing new treatments to improve function. One such method is non-invasive magnetic stimulation (MS) which can modulate the nervous system and may have a role in restoring brain-gut function. The aim of this study is to ascertain whether MS applied to the lumbosacrum can alter human brain gut function, using rectal sensitivity and cortico-anal excitability as our model. Methods: Participants: 13 healthy volunteers (7 females, age range 20-56 years) participated in this study. Sensory measurements were performed via an electrode stimulating catheter positioned in the rectum 10 cm from the anal verge. Sensory and pain thresholds were determined using electrical stimuli [3]. The assessments of sensory and pain thresholds of the rectum were performed at baseline and then immediately, 30 and 60 min after each intervention. Anal sphincter EMG responses were recorded from an anal plug following single pulse transcranial magnetic stimulation over the motor cortex (TMS) and lumbosacrum (LSMS), at baseline and 30 min following each intervention. Interventions comprised 3 different neurostimulation regimens delivered in random order over separate days as repetitive (1 Hz, 10 Hz and sham) lumbosacral magnetic stimulation (rLSMS). Data were (mean±SEM) analysed by two-way ANOVA. Results: Compared to sham, there was a significant increase in sensory and pain thresholds in the rectum in the hour after both 1 Hz rLSMS and 10 Hz rLSMS (*p<0.01, Table 1) By contrast, only 1 Hz rLSMS was accompanied by a significant increase in lumbosacral motor excitability of the anal sphincter (**p<0.05, Table 2). Conclusion: The application of magnetic stimulation to lumbosacral area is able to modulate human visceral sensitivity and corticoanal motor excitability in a frequency dependent manner and holds the promise of being applied to patients with functional GI and defecation disorders as a future therapeutic intervention. Table 1
accuracy of anorectal manometry
1000 Sacral Nerve Stimulation Alters the Frequency of Colon Propagating Sequences in Patients With Neurogenic Fecal Incontinence Vicki E. Patton, John W. Arkwright, David Z. Lubowski, Philip G. Dinning Background: Fecal incontinence (FI) is a chronic condition causing major economic and social burdens. Sacral nerve stimulation (SNS) has emerged as an effective treatment for patients with this condition. However the mode of action by which SNS improves continence remains unclear. Aims: To examine the impact of SNS on colonic propagating sequences (PS) in patients with neurogenic fecal incontinence using our newly developed fibre-optic high resolution manometry catheter in a pilot study in four patients. Methods: Under general
S-161
AGA Abstracts
AGA Abstracts
contraction up to 90μN; (ii) Relaxation of up to 50μN was induced either by Vasoactive Intestinal Peptide (VIP) or 8-bromo-cyclicAMP. Summary: Circular smooth muscle cells maintained viability when cultured on chitosan-coated tissue culture plates and preserved their normal spindle-like shape and expression of contractile smooth muscle markers. Bioengineered tissue constructs harvested from the scaffold maintained their contraction and relaxation responses compared to control tissue constructs that were not placed on the scaffold. Conclusion: Chitosan is non-toxic to colonic smooth muscle cells and provided a good matrix for their survival and maintenance of their contractile phenotype. The tissue constructs maintained the integrity of receptors for VIP and Acetylcholine, and the integrity of intracellular signaling pathways for contraction and relaxation. This is the first report of bioengineering colonic circular smooth muscle tissue constructs on biodegradable chitosan scaffolds. This study provides a basis for creating a functional colon with the possibility of building a longitudinal smooth muscle layer on top of the bioengineered circular layer. Supported by NIH1RC1DK087151.
AGA Abstracts
anesthesia, a fibre-optic manometry catheter (90 recording sites @ 1cm intervals) was positioned colonoscopically, with the tip clipped to the caecum to prevent displacement. A four-electrode tined lead was implanted in 3 patients and a single electrode (Medtronic, Sydney) in one patient, into the S3 sacral nerve foramin and correct placement confirmed by intra-operative xray and nerve stimulation eliciting pelvic floor contraction. After a 2hr basal recording in the fasted state, in a single-blind randomized fashion patients had either sham stimulation or supra-sensory stimulation (14Hz; 300msec). Data are expressed as mean deltas (i.e. stimulation frequency - basal frequency) Results: First we examined the change in frequency of antegrade and retrograde PSs throughout the entire colon during the sham and SNS stimulation. In comparison to sham stimulation, SNS increased the frequency of both antegrade (sham; -2.0 ± 5.5 vs SNS; 9.7 ± 16 PS/2hr) and retrograde (sham; 3.3 ± 6.8 vs SNS; 18 ± 9.1 PS/2hr) propagating sequences. We then looked specifically for changes in the frequency of PSs in the left colon (descending colon and sigmoid), and in comparison to sham stimulation SNS resulted in an increase in the frequency of retrograde PSs (sham; -3 ± 5.6 vs SNS 23 ± 3.6 PS/hr). Conclusion: Sacral nerve stimulation increases both antegrade and retrograde propagating sequences in patients with fecal incontinence. Of potential interest is the increase in retrograde PSs in the distal colon. These data suggest that one mode of action for SNS improving continence is through the alteration of colonic motility.
1003 IGFBP3 Suppresses Reactive Oxygen Species and Cellular Senescence Response to Facilitate TGF-β-Mediated EMT Mitsuteru Natsuizaka, Shinya Ohashi, Azal Ahmadi, Sanders Chang, Kei Nakagawa, Gabrielle S. Wong, Nobuki Miyamoto, Katharine D. Grugan, Andres J. Klein-Szanto, Meenhard Herlyn, J. Alan Diehl, Hiroshi Nakagawa Introduction: Insulin-like growth factor (IGF) binding protein-3 (IGFBP3), a transcriptional target for hypoxia inducible factor (HIF)-1α, is frequently overexpressed in esophageal carcinomas. IGFBP3 facilitates TGF-β-mediated epithelial-to-mesenchymal transition (EMT) in an IGF-independent manner in transformed human esophageal cells (Carcinogenesis. 2010;31:1344-53). Our work indicates that cellular senescence checkpoints need to be suppressed during EMT (Cancer Res. 2010;70:4174-84). We hypothesized that IGFBP3 may alleviate reactive-oxygen species (ROS)-mediated cellular stresses. Methods: Esophageal cancer cell lines and EMT-competent human esophageal epithelial cells transformed by combinations of epidermal growth factor receptor (EGFR) and p53R175H were stably transduced with wild-type (WT) or I56G/L80G/L81G (GGG) mutant IGFBP3, the latter incapable of binding IGFs. Short hairpin RNAs (shRNAs) were used to knockdown IGFBP3. Gene expression was determined by real-time RT-PCR and Western blotting. Senescence-associated β-galactosidase activity was determined. Intracellular ROS level was measured with 2′-7′dichlorodihydrofluorescein diacetate as a fluorescent probe in flow cytometry. Results: Hypoxia (1% O2) induced concomitantly HIF-1α and IGFBP3 in all esophageal cancer cell lines tested (n=10) without eliciting growth inhibitory effects. Interestingly, either hypoxia or exogenous H2O2 induced ROS to a lesser extent in cells expressing a higher level of IGFBP3, implying IGFBP3 in negative regulation of ROS. Upon exposure to hypoxia or H2O2, IGFBP3 knockdown augmented ROS while ectopic expression of either WT or GGGIGFBP3 reduced ROS, indicating an IGF-independent IGFBP3 effect upon ROS generation. Unlike hypoxia, H2O2 reduced cancer cell viability in a dose-dependent manner, and that was augmented by IGFBP3 knockdown and antagonized by ectopic IGFPB3 expression. In EMT-competent transformed cells, TGF-β induced IGFBP3 and EMT, as corroborated by spindle-shaped cell morphology (>80%) and an E-cadherin to N-cadherin class switch, but senescence to a limited extent (<10%). By contrast, TGF-β induced a higher level of ROS and senescence, but not IGFBP3, in empty vector transduced non-transformed control cells that appeared to be less EMT-competent (<20%), where Trolox, a pharmacological antioxidant reduced senescence implying TGF-β-induction of ROS. Conclusions: These mechanistic data suggest that IGFBP3 may have a novel IGF-independent role in the hypoxic tumor microenvironment to alleviate ROS-mediated cellular stresses and senescence to modulate cellular plasticity. This has implications upon EMT in other cancers and may offer platforms for cancer therapy.
1001 CD95 Signaling Induces EMT in Colon Cancer Cells Hanchen Li, Haoxuan Zheng, Jian Hua Liu, Jean Marie Houghton Background The CD95/Fas Ag apoptosis signaling pathway can be used by various cell types for non- apoptotic signaling. Our group has shown that colon cancer cells acquire CD95 apoptosis resistance and gain CD95L expression as they progress from locally invasive disease to metastatic disease. Here we investigate the role of CD95 in the epithelial to mesenchymal transition as cancer cells become invasive. Methods The MC38 colon cancer cell line and the derivatives MC38/FLIP and MC38/FLIP/L have been previously published. The MC38 cell line expresses CD95 on the cell surface and is susceptible to ligand induced apoptosis. MC38/FLIP express the anti-apoptotic protein-FLIP and are resistant to ligand induced apoptosis, but instead proliferate in response to addition of exogenous ligand. MC38/FLIP/ L cells are also resistant to apoptosis however they constitutively express ligand and are more aggressive and proliferate at a higher rate under baseline conditions without the need of additional ligand. We evaluated these cells with and without FasL for the expression of mesenchymal and epithelial specific markers and for EMT specific transcription factors by RT-PCR and Western blot analysis. Results. MC38/FLIP cells assume a more spindle shape in culture when grown in the presence of ligand. Vimentin expression is markedly increased with the addition of ligand. Interestingly, E-cadherin is decreased and N-cadherin expression is markedly increased while B-catenin undergoes relocation to the nucleus with activation of the CD95 pathway in apoptosis resistant colon cancer cells. Conclusion Our findings suggest that in addition to increasing proliferation, CD95 signaling in colon cancer may underlie the EMT important for invasion and metastasis. These findings warn that therapies aimed at inducing apoptosis through classical pathways such as CD95 may need to be approached cautiously, as this pathway may be responsible the aggressive properties of tumors.
1004 Epithelial-to-Mesenchymal Transition and Hematogenous Dissemination Precedes Histologic Diagnosis of Cancer in a Genetic Mouse Model of Pancreatic Ductal Adenocarcinoma Andrew D. Rhim, Ben Stanger BACKGROUND: Less than 20% of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) present with primary tumors of limited size and no clinical evidence of metastasis. Most of these patients undergo pancreatic resection. However, almost all of these patients eventually die of metastatic disease to distant organs. These phenomena suggest that PDAC cell dissemination may precede tumor formation. Here, using a unique and reliable lineage-labeled mouse model of spontaneous PDAC, we sought to determine if epithelial-to-mesenchymal transition (EMT) and hematogenous dissemination occurred at the precancerous pancreatic intraepithelial neoplasm (PanIN) stage. METHODS: We bred Pdx-Cre; LSL-KrasG12D; p53fl/+ or Ink4afl/+; Rosa26LSL-YFP mice in which only pancreatic epithelial cells are permanently labeled with yellow fluorescent protein (YFP). Pancreas and blood specimens from mice aged 2-2.5 mo (mice with only the precancerous PanIN lesion and no histologic evidence of cancer by H&E) and 3.5-4 mo (all with tumors, PDAC) were analyzed. RESULTS: >95% of all pancreatic epithelial cells were labeled with YFP in control Pdx-Cre;RosaYFP mice. Multicolor immunofluorecent (IF) staining for the YFP epithelial lineage label and the mesenchymal markers Zeb1 and FSP1 revealed double positive (EMT+) cells residing in 2.2% and 17.4% of all PanIN2 and PanIN3 lesions, respectively, but in no control Pdx-Cre; RosaYFP mice. Individual spindle-shaped YFP+ cells were observed invading into the pancreatic stroma of PanIN mice, despite the absence of a pathologic diagnosis of cancer based on H&E staining. Using FACS, YFP+ pancreatic cells were found in the blood of both tumor-bearing PDAC mice and PanIN mice (94.5 ± 35.4 v. 42.9 ± 20.5 cells/ml blood; p<0.001; n=30) but not in control Pdx-Cre;RosaYFP mice. These circulating YFP+ cells were devoid of CD45 and Ter-119 (markers of white and red blood cells); expressed YFP, E-cadherin and Pdx-1 transcripts; and formed pancreatospheres in attachment-free cultures. Finally, while rare individual YFP+ cells were found in small hepatic venules of PanIN mice, no metastatic lesions were appreciated, as seen in PDAC mice. CONCLUSIONS: With the use of lineage labeling technology, we provide evidence that EMT, invasion, and hematogenous dissemination may precede histologic diagnosis of PDAC. These data argue against the classical linear progression model of cancer, whereby dissemination occurs after tumors form. However, it is unclear if circulating PanIN cells are the source of metastatic lesions. Nevertheless, based on our data, circulating pancreatic cells may be specific biomarkers for advanced PanIN disease in humans. Further studies to confirm if these data translate to humans are underway.
1002 MTORC1 and mTORC2 Regulate EMT, Motility and Metastasis of Colorectal Cancer via RHOA and RAC1 Signaling Pathways Pat Gulhati, Kanika A. Bowen, Jianyu Liu, Payton D. Stevens, Piotr Rychahou, Min Chen, Eun Y. Lee, Heidi Weiss, Kathleen L. O'Connor, Tianyan Gao, B. M. Evers Activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is associated with growth and progression of colorectal cancer (CRC). The mammalian target of rapamycin (mTOR) kinase acts downstream of PI3K and nucleates two distinct complexes, mTORC1 and mTORC2. The mTOR-RAPTOR complex (mTORC1) regulates translational initiation through the effectors S6K and 4E-BP1. The mTOR-RICTOR complex (mTORC2) regulates the actin cytoskeleton and activates Akt, a key regulator of cell survival. Rapamycin allosterically inhibits the kinase activity of mTORC1, while mTORC2 is believed to be rapamycininsensitive. The contribution of mTOR and its interaction partners in regulating CRC progression and metastasis remains poorly understood. The purpose of the present study was to determine the role of mTORC1 and mTORC2 in regulating EMT, motility and metastasis of CRC. METHODS. HCT116, KM20 and SW480 human colon cancer cells were treated with rapamycin. Cells were also transfected with shRNA directed against RAPTOR or RICTOR. Effects on migration and invasion were analyzed using transwell and wound healing assays. Changes in actin cytoskeleton and cell-cell contacts were determined by immunofluorescence microscopy. RhoA and Rac1 activation was determined using GST-TRBD and GST-PBD pulldown assays, respectively. Establishment of metastasis was determined using intravenous injection of CRC cells. RESULTS. (i) We found that increased expression of mTOR, Raptor and Rictor mRNA was noted with more advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastasis compared to normal colonic tissue. (ii) Treatment with rapamycin significantly decreased the migration and invasion of HCT116, KM20 and SW480 CRC cells. (iii) Stable shRNA-mediated knockdown of Raptor and Rictor significantly decreased the migration and invasion of HCT116, KM20 and SW480 cells. (iv) Stable shRNA-mediated knockdown of Raptor and Rictor leads to mesenchymal-to-epithelial transition and enhances sensitivity to oxaliplatin-induced apoptosis. (v) Stable shRNA-mediated knockdown of Raptor and Rictor leads to characteristic rearrangements in actin cytoskeleton concomitant with decreased RhoA and Rac1 activation. (vi) Establishment of CRC metastasis In Vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. CONCLUSIONS. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating migration, invasion, EMT and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC.
AGA Abstracts
S-162