- Email: [email protected]
547 Intestinal Morphometric and Biomechanical Changes During Aging in Rats
545
ileal segments were measured at the termination of experiment. Morphometric data were obtained by measuring the wall thickness and wall cross-sectional area. The mechanical test was performed as a step-wise distension experiment. The intestinal diameter and length were obtained from digitized images of the segments at pre-selected pressures and at noload and zero-stress states. Circumferential and longitudinal stresses (force per area) and strains (deformation) were computed from the length, diameter and pressure data and from the zero-stress state geometry. Results: The duodenal and ileal dimensions increased slightly from 6 to 22 months, e.g. the weight per unit length, the wall thickness and the wall crosssectional area increased 20%, 4% ,and 25% for duodenum and 12%, 5%, and 8% for ileum, respectively. The opening angle gradually decreased from 154 to 117 degrees for duodenum and from 144 to 87 degrees for ileum as function of aging. The circumferential stress-strain curves shifted to the left after 22 months (p<0.05) whereas the longitudinal stress-strain curves shifted to the left after 18 months (p<0.01) both for duodenum and ileum (Figure 1). The left-shift means that the intestinal wall became stiffer circumferentially and longitudinally following the ageing process. Furthermore, the intestinal wall was stiffer in the longitudinal direction than in the circumferential direction. In conclusion pronounced morphometric and biomechanical remodeling occurred in the rat intestine during aging. The observed changes likely reflect the changes of the physiological function of the intestine during ageing, similar to other tissues where function, mechanical loading and form are interrelated factors. Keywords: intestine; morphometry; residual strain; opening angle; stress-strain; aging.
AGA Abstracts
BMP4 Signaling Is Involved in Epithelial-Mesenchymal Transition in Barrett's Esophagus and Esophageal Adenocarcinoma Through Induction of SNAIL2 Christine Kestens, Peter D. Siersema, Jantine W. van Baal BACKGROUND: Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett's esophagus (BE), a premalignant condition of the esophagus with an increased risk of developing esophageal adenocarcinoma (EAC). In pancreatic and ovarian cancer, BMP4 has been found to induce epithelial mesenchymal transition (EMT) by inducing the transcription factor SNAIL2 and subsequently reducing CDH1. Its function in the development of EAC is however unclear. AIM: To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally to determine the effect of BMP4 signaling on EMT in a human Barrett's esophagus (BAR-T) and adenocarcinoma (OE-33) cell line. METHODS: Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR and Western blot analysis using biopsy samples of 10 normal squamous esophagus (SQ), 10 BE and 10 EAC patients. BAR-T and OE-33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of EMT markers (SNAIL2, CDH1 and CDH2) were evaluated by Q-RT-PCR and Western Blot analysis. RESULTS: Compared to SQ, BMP4 expression was 34.2-fold increased in EAC and 32.7-fold in BE (p<0.01) The expression of the downstream target, ID2 was 10.5-fold increased in EAC and 7.4-fold in BE compared to SQ (p<0.01). SMAD4 expression was 3.0-fold increased in EAC and 2.6-fold in BE compared to SQ (p<0.01). BMP receptor (BMPR) 1a, BMPR1b, BMPR2, SMAD1 and SMAD5 were all expressed and did not show a significant difference between the various tissues. Western blot analysis confirmed our results, showing an increased expression of BMP4, pSMAD1/5/8 and ID2 in both BE and EAC. BMP4 incubation inhibited cell viability, however induced cell migration in both BAR-T and OE-33 cells. These effects were not observed when cells were incubated with the BMP4 antagonist, Noggin. Upon BMP4 incubation, SNAIL2 expression was significantly increased in BAR-T and OE-33 cells (1.6-fold and 3.6-fold, respectively) and CDH1 expression was significantly decreased (0.6fold in both cell lines). Additionally CDH2 expression was increased in both BAR-T (2.7fold) and OE-33 cells (12.5-fold) incubated with BMP4. These results were confirmed by Western Blot analysis. CONCLUSION: Our results indicate active BMP4 signaling in BE and EAC. This may result in an increased malignant phenotype by inducing EMT through increased expression of the transcription factor SNAIL2, which subsequently represses CDH1 expression and induces CDH2 expression. Future manipulation of the BMP4 pathway may help to prevent neoplastic progression in the esophagus.
546 Nerve Growth Factor (NGF) Deficiency in Aging Gastric Mucosa: A New, Previously Unrecognized Mechanism of Aging-Related Impairment of Gastric Angiogenesis. NGF Therapy Completely Reverses This Impairment Amrita Ahluwalia, Michael K. Jones, Tomasz Brzozowski, Andrzej S. Tarnawski
Figure 1: Circumferential and longitudinal stress-strain in different age groups. For the circumferential direction, the curves shifted to the left at age 22 months. For the longitudinal direction the curves shifted to the left at age 18 months. Left-shift indicates wall stiffening. Both for duodenum and ileum, the longitudinal stress-strain curves were located to the left of the circumferential stress-strain curves, demonstrating the highest stiffness in the longitudinal direction.
Background/Aims: Angiogenesis in gastric mucosa and gastric endothelial cells (ECs) is impaired in aging; however, the mechanisms are not fully understood. Nerve growth factor (NGF, originally discovered as a factor critical for neuron survival) has been shown to induce angiogenesis in some ECs, e.g. chorioidal ECs. The expression of NGF in gastric ECs, its alteration in aging and its role in gastric angiogenesis are not known. We studied in gastric mucosal ECs (GMECs) isolated from young and aging rats: (1) expression of NGF and its high affinity receptor, TrkA; (2) in vitro angiogenesis and (3) whether treatment of aging GMECs with exogenous NGF or NGF overexpression could reverse impairment of angiogenesis in these cells. Methods: GMECs were isolated from Fisher F-344 rats, 3 month of age (young, YGMEC) and 24 month of age (aging, AGMEC) using anti-PECAM-1 selection and magnetic bead separation. YGMECs and AGMECs were treated with either: vehicle, NGF (10-100 ng/ml), VEGF (10-100 ng/ml); or, were transduced with lentiviral (LV)-NGF (NGF gene therapy) or LV-GFP (control). Studies: 1) NGF and TrkA expression by Western blotting and immunohistochemistry; 2) In vitro angiogenesis on matrigel and quantification of vascular tube formation at 6 and 24 hours using a Metamorph 7.0 image analysis system; 3) EC proliferation by BrdU assay;and, 4) F-actin cytoskeletal rearrangements in ECs. Results: In AGMECs, expression of NGF and TrkA was ~3.5-fold lower (P<0.01) vs. YGMECs and in vitro angiogenesis was significantly reduced by 3.6-fold (P<0.001). Treatment of AGMECs with exogenous NGF or gene therpay using LV-NGF significantly increased in vitro angiogenesis by 1.8- and 3.7-fold, respectively (both P<0.001). This increase was further enhanced by co-treatment with VEGF. LV-NGF transduction of AGMECs (gene therapy) completely reversed aging-related angiogenesis impairment in these cells, caused a 38% increase in cell proliferation and induced formation of extensive, long filopodia reflecting a change in these cells to an angiogenic phenotype. Moreover, the filopodia demonstrated a strong NGF expression indicating NGF binding to F-actin bundles. Conclusions: This study demonstrates for the first time that: 1) Gastric ECs express NGF and its receptor, TrkA; 2) NGF and TrkA expression is significantly reduced in aging GMECs (vs. young) and strongly correlates with impaired angiogenesis; 3) Exogenous NGF or NGF gene therapy significantly increases in vitro angiogenesis and cell proliferation in aging GMECs demonstrating a causal role of NGF in gastric angiogenesis and its impairment in aging. This study uncovered a novel role for NGF in GMECs, gastric angiogenesis and its aging-related impairment, and this paradigm may also apply to other aging tissues.
548 Gastrointestinal (GI) Findings in Patients With Biallelic Mismatch Repair (BMMRD) Gene Deficiency Syndrome: Report From the International Consortium Carol Durno, Melyssa Aronson, Uri Tabori, Steven Gallinger, Shlomi Cohen, Rina Dvir, Ronit Elhasid, Hagit B. Feldman, Harriet Druker, Helen Chan, Simon Ling, Paul P. Kortan, Spring Holter, Kara Semotiuk, David Malkin, Roula Farah, Brandie Leach, Matthew F. Kalady, Lynette S. Penney, Andrea L. Rideout, Mohsin Rashid, Linda Hasadsri, Pavel Pichurin, Douglas Riegert-Johnson, Brittany Campbell, Doua Bakry, Hala Rimawi, Qasim K. Alharbi, Musa M. Alharbi, Shamvil Ashraf, Revital Kariv, Alain Sayad, Zane Cohen Background: BMMRD causes a novel syndrome with a distinct phenotype from classic Lynch syndrome. BMMRD individuals manifest features of neurofibromatosis type 1 (NF1), and they also develop brain tumors and hematological cancers. The gastrointestinal phenotype includes polyposis and early onset gastrointestinal cancer. Objective: To prospectively characterize the gastrointestinal phenotype in a large cohort of BMMRD patients using surveillance data gathered to date to determine age of onset of polyps, prevalence of polyps and cancer, anatomical distribution of cancer, colorectal surgical intervention and outcome, as well as polyp progression in BMMRD. Methods: The international BMMRD consortium was created in 2010 to collect clinical, genetic and outcome information. Demographic, genetic and medical information are obtained on all BMMRD families and stored at the Zane Cohen Centre's Familial GI Cancer Registry at Mount Sinai Hospital, and the Hospital for Sick Children, Toronto, Canada. Patients were included after validation of germline biallelic mutations and/or negative immunohistochemical staining for the corresponding mismatch repair gene in normal and cancerous tissue. GI screening data including endoscopy, surgical and pathology reports were collected on all participants with yearly updates. GI neoplasms were confirmed through medical records. Results: Thirty-five individuals (19 kindreds), from 7 countries were identified with BMMRD. Gastrointestinal evaluation and surveillance data was available on 24 of 33 (73%) individuals who were of age to screen totaling fiftythree patient years. Eight patients had 9 invasive adenocarcinomas (median 16.7 years, range 8-25). Three patients had synchronous colorectal cancer (3, 4, 6 tumors). Small bowel adenocarcinoma was diagnosed in 4 patients (median 18 years, range 11-33). The youngest patient with colonic polyps was 8 years. The number of polyps ranged from 1 to 100. High grade dysplasia was identified at a median of 11 years (range 8-11 years). 100% of patients had colonic polyps or colonic adenocarcinoma by 18 years. The youngest patient with a small bowel polyp was 11 years of age. Among patients undergoing surveillance GI mortality has been zero. Four patients died of a non-GI cancer. 96% of affected patients had caféau-lait macules. Consanguinity was identified in 14 kindreds (58%). Conclusions: The
547 Intestinal Morphometric and Biomechanical Changes During Aging in Rats Jingbo Zhao, Hans Gregersen Background and aim: Previously we demonstrated pronounced morphometric and biomechanical remodeling in the rat intestine during physiological growth up to 32 weeks of age. The aim of the present study is to study intestinal geometric and biomechanical changes in aging rats. Materials and methods: Twenty-four male Wistar rats, aged from 6 to 22 months, were used in the study. The body weight and the wet weight per length of duodenal and
AGA Abstracts
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ileal segments were measured at the termination of experiment. Morphometric data were obtained by measuring the wall thickness and wall cross-sectional area. The mechanical test was performed as a step-wise distension experiment. The intestinal diameter and length were obtained from digitized images of the segments at pre-selected pressures and at noload and zero-stress states. Circumferential and longitudinal stresses (force per area) and strains (deformation) were computed from the length, diameter and pressure data and from the zero-stress state geometry. Results: The duodenal and ileal dimensions increased slightly from 6 to 22 months, e.g. the weight per unit length, the wall thickness and the wall crosssectional area increased 20%, 4% ,and 25% for duodenum and 12%, 5%, and 8% for ileum, respectively. The opening angle gradually decreased from 154 to 117 degrees for duodenum and from 144 to 87 degrees for ileum as function of aging. The circumferential stress-strain curves shifted to the left after 22 months (p<0.05) whereas the longitudinal stress-strain curves shifted to the left after 18 months (p<0.01) both for duodenum and ileum (Figure 1). The left-shift means that the intestinal wall became stiffer circumferentially and longitudinally following the ageing process. Furthermore, the intestinal wall was stiffer in the longitudinal direction than in the circumferential direction. In conclusion pronounced morphometric and biomechanical remodeling occurred in the rat intestine during aging. The observed changes likely reflect the changes of the physiological function of the intestine during ageing, similar to other tissues where function, mechanical loading and form are interrelated factors. Keywords: intestine; morphometry; residual strain; opening angle; stress-strain; aging.
AGA Abstracts
BMP4 Signaling Is Involved in Epithelial-Mesenchymal Transition in Barrett's Esophagus and Esophageal Adenocarcinoma Through Induction of SNAIL2 Christine Kestens, Peter D. Siersema, Jantine W. van Baal BACKGROUND: Bone morphogenetic protein 4 (BMP4) signaling is involved in the development of Barrett's esophagus (BE), a premalignant condition of the esophagus with an increased risk of developing esophageal adenocarcinoma (EAC). In pancreatic and ovarian cancer, BMP4 has been found to induce epithelial mesenchymal transition (EMT) by inducing the transcription factor SNAIL2 and subsequently reducing CDH1. Its function in the development of EAC is however unclear. AIM: To investigate the expression of BMP4 and several members of the BMP4 pathway in EAC. Additionally to determine the effect of BMP4 signaling on EMT in a human Barrett's esophagus (BAR-T) and adenocarcinoma (OE-33) cell line. METHODS: Expression of BMP4, its downstream target ID2 and members of the BMP4 pathway were determined by Q-RT-PCR and Western blot analysis using biopsy samples of 10 normal squamous esophagus (SQ), 10 BE and 10 EAC patients. BAR-T and OE-33 cells were incubated with BMP4 or the BMP4 antagonist, Noggin, and cell viability and migration assays were performed. In addition, expression of EMT markers (SNAIL2, CDH1 and CDH2) were evaluated by Q-RT-PCR and Western Blot analysis. RESULTS: Compared to SQ, BMP4 expression was 34.2-fold increased in EAC and 32.7-fold in BE (p<0.01) The expression of the downstream target, ID2 was 10.5-fold increased in EAC and 7.4-fold in BE compared to SQ (p<0.01). SMAD4 expression was 3.0-fold increased in EAC and 2.6-fold in BE compared to SQ (p<0.01). BMP receptor (BMPR) 1a, BMPR1b, BMPR2, SMAD1 and SMAD5 were all expressed and did not show a significant difference between the various tissues. Western blot analysis confirmed our results, showing an increased expression of BMP4, pSMAD1/5/8 and ID2 in both BE and EAC. BMP4 incubation inhibited cell viability, however induced cell migration in both BAR-T and OE-33 cells. These effects were not observed when cells were incubated with the BMP4 antagonist, Noggin. Upon BMP4 incubation, SNAIL2 expression was significantly increased in BAR-T and OE-33 cells (1.6-fold and 3.6-fold, respectively) and CDH1 expression was significantly decreased (0.6fold in both cell lines). Additionally CDH2 expression was increased in both BAR-T (2.7fold) and OE-33 cells (12.5-fold) incubated with BMP4. These results were confirmed by Western Blot analysis. CONCLUSION: Our results indicate active BMP4 signaling in BE and EAC. This may result in an increased malignant phenotype by inducing EMT through increased expression of the transcription factor SNAIL2, which subsequently represses CDH1 expression and induces CDH2 expression. Future manipulation of the BMP4 pathway may help to prevent neoplastic progression in the esophagus.
546 Nerve Growth Factor (NGF) Deficiency in Aging Gastric Mucosa: A New, Previously Unrecognized Mechanism of Aging-Related Impairment of Gastric Angiogenesis. NGF Therapy Completely Reverses This Impairment Amrita Ahluwalia, Michael K. Jones, Tomasz Brzozowski, Andrzej S. Tarnawski
Figure 1: Circumferential and longitudinal stress-strain in different age groups. For the circumferential direction, the curves shifted to the left at age 22 months. For the longitudinal direction the curves shifted to the left at age 18 months. Left-shift indicates wall stiffening. Both for duodenum and ileum, the longitudinal stress-strain curves were located to the left of the circumferential stress-strain curves, demonstrating the highest stiffness in the longitudinal direction.
Background/Aims: Angiogenesis in gastric mucosa and gastric endothelial cells (ECs) is impaired in aging; however, the mechanisms are not fully understood. Nerve growth factor (NGF, originally discovered as a factor critical for neuron survival) has been shown to induce angiogenesis in some ECs, e.g. chorioidal ECs. The expression of NGF in gastric ECs, its alteration in aging and its role in gastric angiogenesis are not known. We studied in gastric mucosal ECs (GMECs) isolated from young and aging rats: (1) expression of NGF and its high affinity receptor, TrkA; (2) in vitro angiogenesis and (3) whether treatment of aging GMECs with exogenous NGF or NGF overexpression could reverse impairment of angiogenesis in these cells. Methods: GMECs were isolated from Fisher F-344 rats, 3 month of age (young, YGMEC) and 24 month of age (aging, AGMEC) using anti-PECAM-1 selection and magnetic bead separation. YGMECs and AGMECs were treated with either: vehicle, NGF (10-100 ng/ml), VEGF (10-100 ng/ml); or, were transduced with lentiviral (LV)-NGF (NGF gene therapy) or LV-GFP (control). Studies: 1) NGF and TrkA expression by Western blotting and immunohistochemistry; 2) In vitro angiogenesis on matrigel and quantification of vascular tube formation at 6 and 24 hours using a Metamorph 7.0 image analysis system; 3) EC proliferation by BrdU assay;and, 4) F-actin cytoskeletal rearrangements in ECs. Results: In AGMECs, expression of NGF and TrkA was ~3.5-fold lower (P<0.01) vs. YGMECs and in vitro angiogenesis was significantly reduced by 3.6-fold (P<0.001). Treatment of AGMECs with exogenous NGF or gene therpay using LV-NGF significantly increased in vitro angiogenesis by 1.8- and 3.7-fold, respectively (both P<0.001). This increase was further enhanced by co-treatment with VEGF. LV-NGF transduction of AGMECs (gene therapy) completely reversed aging-related angiogenesis impairment in these cells, caused a 38% increase in cell proliferation and induced formation of extensive, long filopodia reflecting a change in these cells to an angiogenic phenotype. Moreover, the filopodia demonstrated a strong NGF expression indicating NGF binding to F-actin bundles. Conclusions: This study demonstrates for the first time that: 1) Gastric ECs express NGF and its receptor, TrkA; 2) NGF and TrkA expression is significantly reduced in aging GMECs (vs. young) and strongly correlates with impaired angiogenesis; 3) Exogenous NGF or NGF gene therapy significantly increases in vitro angiogenesis and cell proliferation in aging GMECs demonstrating a causal role of NGF in gastric angiogenesis and its impairment in aging. This study uncovered a novel role for NGF in GMECs, gastric angiogenesis and its aging-related impairment, and this paradigm may also apply to other aging tissues.
548 Gastrointestinal (GI) Findings in Patients With Biallelic Mismatch Repair (BMMRD) Gene Deficiency Syndrome: Report From the International Consortium Carol Durno, Melyssa Aronson, Uri Tabori, Steven Gallinger, Shlomi Cohen, Rina Dvir, Ronit Elhasid, Hagit B. Feldman, Harriet Druker, Helen Chan, Simon Ling, Paul P. Kortan, Spring Holter, Kara Semotiuk, David Malkin, Roula Farah, Brandie Leach, Matthew F. Kalady, Lynette S. Penney, Andrea L. Rideout, Mohsin Rashid, Linda Hasadsri, Pavel Pichurin, Douglas Riegert-Johnson, Brittany Campbell, Doua Bakry, Hala Rimawi, Qasim K. Alharbi, Musa M. Alharbi, Shamvil Ashraf, Revital Kariv, Alain Sayad, Zane Cohen Background: BMMRD causes a novel syndrome with a distinct phenotype from classic Lynch syndrome. BMMRD individuals manifest features of neurofibromatosis type 1 (NF1), and they also develop brain tumors and hematological cancers. The gastrointestinal phenotype includes polyposis and early onset gastrointestinal cancer. Objective: To prospectively characterize the gastrointestinal phenotype in a large cohort of BMMRD patients using surveillance data gathered to date to determine age of onset of polyps, prevalence of polyps and cancer, anatomical distribution of cancer, colorectal surgical intervention and outcome, as well as polyp progression in BMMRD. Methods: The international BMMRD consortium was created in 2010 to collect clinical, genetic and outcome information. Demographic, genetic and medical information are obtained on all BMMRD families and stored at the Zane Cohen Centre's Familial GI Cancer Registry at Mount Sinai Hospital, and the Hospital for Sick Children, Toronto, Canada. Patients were included after validation of germline biallelic mutations and/or negative immunohistochemical staining for the corresponding mismatch repair gene in normal and cancerous tissue. GI screening data including endoscopy, surgical and pathology reports were collected on all participants with yearly updates. GI neoplasms were confirmed through medical records. Results: Thirty-five individuals (19 kindreds), from 7 countries were identified with BMMRD. Gastrointestinal evaluation and surveillance data was available on 24 of 33 (73%) individuals who were of age to screen totaling fiftythree patient years. Eight patients had 9 invasive adenocarcinomas (median 16.7 years, range 8-25). Three patients had synchronous colorectal cancer (3, 4, 6 tumors). Small bowel adenocarcinoma was diagnosed in 4 patients (median 18 years, range 11-33). The youngest patient with colonic polyps was 8 years. The number of polyps ranged from 1 to 100. High grade dysplasia was identified at a median of 11 years (range 8-11 years). 100% of patients had colonic polyps or colonic adenocarcinoma by 18 years. The youngest patient with a small bowel polyp was 11 years of age. Among patients undergoing surveillance GI mortality has been zero. Four patients died of a non-GI cancer. 96% of affected patients had caféau-lait macules. Consanguinity was identified in 14 kindreds (58%). Conclusions: The
547 Intestinal Morphometric and Biomechanical Changes During Aging in Rats Jingbo Zhao, Hans Gregersen Background and aim: Previously we demonstrated pronounced morphometric and biomechanical remodeling in the rat intestine during physiological growth up to 32 weeks of age. The aim of the present study is to study intestinal geometric and biomechanical changes in aging rats. Materials and methods: Twenty-four male Wistar rats, aged from 6 to 22 months, were used in the study. The body weight and the wet weight per length of duodenal and
AGA Abstracts
X : 55624$1AGA 03-28-15 04:55:56 PDFd : 55624B : e
S-110
Page 110