Vascular endothelial growth factor C (VEGF-C) in esophageal ...
Sa1935 The Effect of Diabetes Mellitus on Lower Esophageal Sphincter Muscle and Esophageal Epithelia...
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AGA Abstracts clarithromycin (1μg/ml). All these 7 clarithromycin-resistant strains had the BsaI site (Figure 1). We have found mutations site at A1825G, T1834C and A2147G mutation (Figure 2). None had the new BbsI cleavage site which is characteristic of the other relatively common mutation (A2143G). The sequence of the 23S rRNA gene of the ClaR strain no 34A has been submitted and the accession No is JX827460. We introduced the PCR fragments containing the relevant mutations amplified with primer pair as listed above from the chromosomal DNA of strain 3B (ClaS strain) into H. pylori 34 and 39 (ClaR strain). The relevant mutations in both transformants were confirmed by DNA sequencing to be the same as those in the donor strain. By examining their effects in an isogenic background, it was found that the MIC of clarithromycin was the same for both transformants (4 μg/ml). Conclusion: Our results show an increasing trend of h.pylori towards dual resistance with metronidazole and clarithromycin, but maintained sensitivity towards tetracycline and amoxi- cillin. Clarithromycin resistance was due to mutations at site A1825G, T1834C and A2147G of 23 S rRNA gene. Figure 1: RFLP of representive clarithromycin resistant strains. Lane 1 clarithromycin sensitive strain 3B, Lane 2 to 5 are claR strains, lane 6 sentive strain 26695 and lane N is negative control Figure 2: Representative sequence showing A to G mutation at position 2147 of 23S rRNA gene. Sa1933 Reduced Expression of Nerve Growth Factor and Its Receptors in Scars of Healed Gastric Ulcers: A Major Mechanism Underlying Impaired Neural Plexus and Sensory Nerve Regeneration in Ulcer Scars and a Novel Causal Role in Abnormal Neovascularization? Andrzej S. Tarnawski, Amrita Ahluwalia, Michael K. Jones, Tamara Matysiak-Budnik Background/Aims: Scars of healed gastric ulcers (GU) exhibit prominent epithelial and vascular abnormalities. Whether regeneration of submucosal neural plexus and sensory nerves is also impaired has not been fully explored. This study was aimed to test the hypothesis that regeneration of neural plexuses and sensory nerves in GU scars is impaired due to sustained local downregulation of the fundamental neurotrophic factor, nerve growth factor (NGF), and/or its receptors, TrkA and p75NTR. Methods: GU were induced in rats by focal serosal application of acetic acid. Gastric wall specimens containing ulcers or ulcer scars were obtained 3, 7 and 14 days and 3 months post ulcer induction. Studies: 1) Quantitative histology, 2) vascular casts using Mercox resin + SEM, 3) gene microarray analysis (Affymetrix chips ~7,000 genes), 4) visualization of neuronal cells and nerve fibers using a specific neuronal marker (HuC), 5) expression of NGF, TrkA and p75NTR by Western blotting and immunochemistry; and, 6) expression of the sensory nerve marker, calcitonin gene related peptide (CGRP). To determine clinical relevance, 21 specimens of human GU and scars were examined for regeneration of submucosal and myenteric plexuses, NGF expression and presence of CGRP+ sensory nerves. Results: In normal rat gastric mucosa HuC staining visualized neuronal cells and plexuses. CGRP+ nerves were distributed along blood vessels and mucosal microvessels. NGF was strongly expressed in: a) neural ganglion and Schwann's cells and nerves of the submucosal plexus, b) epithelial cells lining gastric glands; and, c) endothelial cells of submucosal blood vessels. In GU scars, gastric glands were not fully restored and blood microvessels were abnormal, reflecting impaired and incomplete neovas- cularization. There was minimal or no regeneration of neural plexuses; and, CGRP+ sensory nerves were reduced by .40-fold; p,0.001 vs. normal mucosa or submucosa. In GU scars expression of NGF mRNA was reduced .5-fold; NGF, TrkA and p75 NTR proteins were reduced by 6 - 15-fold (all p ,0.001 vs. normal mucosa or submucosa). NGF expression in epithelial, neural and endothelial cells of GU scar was minimal or absent. Scars of human GU showed similar findings. Conclusions:1) Scars of healed GU exhibit: (a) impaired regeneration of neural elements and (b) reduced expression of NGF and its receptors. 2) Since NGF promotes survival of neuronal cells and stimulates axonal regeneration, its reduced expression likely results in impaired regeneration of neural plexuses and sensory nerves in GU scars. 3) Because NGF can also regulate neovascularization, local NGF deficiency may underlie abnormal microvessel regeneration in GU scars. 4) This study highlights the interde- pendence of epithelial, neural and endothelial cells; all of which generate NGF in normal gastric mucosa and interact during GU healing. S-338 AGA Abstracts Sa1934 Importance of Thromboxane A2/Prostacyclin Balance in Pathogenic Mechanism of Ischemia/ Reperfusion-Induced Gastric Injury in Mice Koji Takeuchi, Yuka Nakamori, Shinji Kojima, Tohru Kotani Background/Aim: Eicosanoids are involved in modulating the gastric mucosal integrity under various conditions. We previously reported that cyclooxygenase (COX)-2/ prostacyclin (PGI2) play a crucial role in the gastric mucosal defense under ischemia/ reperfusion (I/R)- induced conditions, whereas indomethacin worsens these lesions, at least partly, through the up-regulation of cysteinyl leukotrienes (CysLTs) and their receptors. In the present study, we examined the role of thromboxane A2 (TXA2) in the ulcerogenic response in the mouse stomach under I/R-induced conditions. Methods: Male C57BL/6J mice were used after 18 h fasting. Under urethane anesthesia, the celiac artery was clamped (ischemia) for 30 min, followed by reperfusion by removal of the clamp. After reperfusion for 60 min, the area of macroscopically visible lesions in the stomach was measured. Indomethacin (5 mg/ kg) was administered SC 30 min before the onset of ischemia. SC-560 (COX-1 inhibitor: 5 mg/kg), rofecoxib (COX-2 inhibitor: 5 mg/kg), ozagrel (TXA2 synthase inhibitor: 200 mg/kg), seratrodast (TXA2 antagonist: 1-30 mg/kg) or pranlukast (CysLT receptor type 1 antagonist: 10 mg/kg) was administered PO 60 min before ischemia. Iloprost (1 μg/kg) was given IV 5 min before reperfusion. TXB2 and 6-keto-PGF1 α contents were measured by enzyme immunoassay. The gene expressions of TP receptor and TXA2 synthase were exam- ined by RT-PCR. Results: I/R produced hemorrhagic lesions in the gastric mucosa, and the severity of these lesions was reduced by pretreatment with ozagrel, seratrodast or iloprost, in addition to pranlukast. By contrast, the gastric ulcerogenic response under I/R conditions was worsened by pretreatment with indomethacin and rofecoxib but not SC-560, and this aggravation was both abrogated by co-administration of iloprost. Pretreatment of ozagrel or seratrodast attenuated the worsening effect of rofecoxib but not the effect of indomethacin that was abrogated by pranlukast. I/R significantly increased both TXB2 and 6-keto-PGF1a contents in the gastric mucosa. The increase of TXB2 content under I/R conditions was attenuated by SC-560, while the increased PGI2 biosynthetic response was totally attenuated by rofecoxib. I/R by itself had no effect on the TXB2/ 6-keto-PGF1a ratio in the gastric mucosa, yet the ratio was markedly increased by rofecoxib and decreased by ozagrel, respectively. The gene expressions of TP receptor and TXA2 synthase were observed in the normal stomach and remained unchanged under I/R-induced conditions. Conclusion: These results suggest that 1) COX-1/TXA2 plays a crucial role in the development of I/R-induced gastric lesions, in addition to COX-2/PGI2 as well as CysLTs, and 2) the aggravation by rofecoxib of I/R-induced gastric lesions may be accounted partly for by the imbalance of TXA2/PGI2 ratio in the stomach. Sa1935 The Effect of Diabetes Mellitus on Lower Esophageal Sphincter Muscle and Esophageal Epithelia in Rabbit Doga Capanoglu, Deniz Coskunsever, Murat Olukman, Serhat Bor Gastrointestinal motility disorders were commonly seen in patients with Diabetes mellitus (DM). A weak relationship was reported between DM-GERD and with esophageal motility disturbances. The possible mechanisms are not known except neuropathy. We aimed to investigate the electrophysiological changes of diabetic rabbit esophageal epithelia (EE) and lower esophageal sphincter (LES) muscle using Ussing chamber system and isolated organ bath. METHOD: Chronic diabetes in New Zealand white rabbits were created using alloxane (100mg/kg). Blood sugar levels were measured 72 hours after drug administration, values over 250mg/dl was accepted as diabetic. Rabbits were sacrificed at 8th week; EE was placed on Ussing chambers. Potential difference (PD), short circuit current (Isc) were measured, electrical resistance (R) was calculated. Acid-pepsin model was employed by titrating the ringer solution of luminal side of the tissues to acidic (pH 1.7) or weakly acidic (pH 4) and/or addition of pepsin (0.25mg/ml). Tissues were fixed for morphological evaluation. Smooth muscle strips taken from LES were mounted to isolated organ bath, and kept in resting tension for 1 hour. Electrical Field Stimulation (EFS) and acetyl choline cumulative muscle contraction response were recorded. These muscle contraction responses were also tested in the presence of Rho-kinase inhibitor (Y-27632) and nonspecific nitric oxide inhibitor (L-NAME). RESULTS: The PD of diabetic tissues perfused in acidified ringer+pepsin dropped 15%, while control tissues dropped 40%. Diabetic tissue resistance perfused in acidified ringer solution decreased 10%, pepsin addition decreased 47%. Same concentrations caused a more distinct loss of resistance in control tissues (26% and 78%) (p ,0.05 compared to control group). EFS responses was found to increase in diabetic group compared to control (p,0,001). Y-27632 and L-NAME was found to increase contraction response with higher concentration without any statistical significance. The response increased with acetyl choline in diabetic group with cumulative concentrations (p ,0,001). In diabetic group; increased concentrations of Y-27632 was found to significantly decreased acetyl choline responses. This effect was not observed in control group. Histopathologic scores were the same between groups. DISCUSSION: Even though acid-pepsin model causes mucosal damage, diabetic rabbit esophageal tissues had less injury compared to control. The isolated organ bath studies supported these findings; in diabetic rabbits, LES contraction response increased significantly, and Rho-Rhokinase pathway is found to be responsible of this change. These results can be interpreted that, in chronic diabetic rabbit model, esophagus was found to resist against reflux by mucosal defence mechanisms and increased contraction pressures. Sa1936 Unique Protective Effect of Egualen, a Stable Azulene Derivative, on Gastrointestinal Lesions Induced in Rats by Ischemia/Reperfusion, Double Antithrombotic Therapy, and Loxoprofen Kikuko Amagase, Yuji Yoshida, Daisuke Hara, Toshiko Murakami, Koji Takeuchi Background/Aim: Egualen Na (3-ethyl-7-isopropyl-1-azulemesulfonate 1/3 hydrate Na), a stable azulene derivative, has been used as a mucosal protective drug for the treatment of gastritis and gastric ulcers in Japan. This agent exhibits antiulcer activity against a variety
Transcript of Sa1935 The Effect of Diabetes Mellitus on Lower Esophageal Sphincter Muscle and Esophageal Epithelia...
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sclarithromycin (1μg/ml). All these 7 clarithromycin-resistant strains had the BsaI site (Figure1). We have found mutations site at A1825G, T1834C and A2147G mutation (Figure 2).None had the new BbsI cleavage site which is characteristic of the other relatively commonmutation (A2143G). The sequence of the 23S rRNA gene of the ClaR strain no 34A hasbeen submitted and the accession No is JX827460. We introduced the PCR fragmentscontaining the relevant mutations amplified with primer pair as listed above from thechromosomal DNA of strain 3B (ClaS strain) into H. pylori 34 and 39 (ClaR strain). Therelevant mutations in both transformants were confirmed by DNA sequencing to be thesame as those in the donor strain. By examining their effects in an isogenic background, itwas found that the MIC of clarithromycin was the same for both transformants (4 μg/ml).Conclusion: Our results show an increasing trend of h.pylori towards dual resistance withmetronidazole and clarithromycin, but maintained sensitivity towards tetracycline and amoxi-cillin. Clarithromycin resistance was due to mutations at site A1825G, T1834C and A2147Gof 23 S rRNA gene.
Figure 1: RFLP of representive clarithromycin resistant strains. Lane 1 clarithromycin sensitivestrain 3B, Lane 2 to 5 are claR strains, lane 6 sentive strain 26695 and lane N is negative control
Figure 2: Representative sequence showing A to G mutation at position 2147 of 23SrRNA gene.
Sa1933
Reduced Expression of Nerve Growth Factor and Its Receptors in Scars ofHealed Gastric Ulcers: A Major Mechanism Underlying Impaired NeuralPlexus and Sensory Nerve Regeneration in Ulcer Scars and a Novel CausalRole in Abnormal Neovascularization?Andrzej S. Tarnawski, Amrita Ahluwalia, Michael K. Jones, Tamara Matysiak-Budnik
Background/Aims: Scars of healed gastric ulcers (GU) exhibit prominent epithelial and vascularabnormalities. Whether regeneration of submucosal neural plexus and sensory nerves is alsoimpaired has not been fully explored. This study was aimed to test the hypothesis thatregeneration of neural plexuses and sensory nerves in GU scars is impaired due to sustainedlocal downregulation of the fundamental neurotrophic factor, nerve growth factor (NGF),and/or its receptors, TrkA and p75NTR. Methods: GU were induced in rats by focal serosalapplication of acetic acid. Gastric wall specimens containing ulcers or ulcer scars wereobtained 3, 7 and 14 days and 3 months post ulcer induction. Studies: 1) Quantitativehistology, 2) vascular casts using Mercox resin + SEM, 3) gene microarray analysis (Affymetrixchips ~7,000 genes), 4) visualization of neuronal cells and nerve fibers using a specificneuronal marker (HuC), 5) expression of NGF, TrkA and p75NTR by Western blotting andimmunochemistry; and, 6) expression of the sensory nerve marker, calcitonin gene relatedpeptide (CGRP). To determine clinical relevance, 21 specimens of human GU and scarswere examined for regeneration of submucosal and myenteric plexuses, NGF expressionand presence of CGRP+ sensory nerves. Results: In normal rat gastric mucosa HuC stainingvisualized neuronal cells and plexuses. CGRP+ nerves were distributed along blood vesselsand mucosal microvessels. NGF was strongly expressed in: a) neural ganglion and Schwann'scells and nerves of the submucosal plexus, b) epithelial cells lining gastric glands; and, c)endothelial cells of submucosal blood vessels. In GU scars, gastric glands were not fullyrestored and blood microvessels were abnormal, reflecting impaired and incomplete neovas-cularization. There was minimal or no regeneration of neural plexuses; and, CGRP+ sensorynerves were reduced by .40-fold; p,0.001 vs. normal mucosa or submucosa. In GU scarsexpression of NGF mRNA was reduced .5-fold; NGF, TrkA and p75 NTR proteins werereduced by 6 - 15-fold (all p ,0.001 vs. normal mucosa or submucosa). NGF expressionin epithelial, neural and endothelial cells of GU scar was minimal or absent. Scars ofhuman GU showed similar findings. Conclusions:1) Scars of healed GU exhibit: (a) impairedregeneration of neural elements and (b) reduced expression of NGF and its receptors. 2)Since NGF promotes survival of neuronal cells and stimulates axonal regeneration, its reducedexpression likely results in impaired regeneration of neural plexuses and sensory nerves inGU scars. 3) Because NGF can also regulate neovascularization, local NGF deficiency mayunderlie abnormal microvessel regeneration in GU scars. 4) This study highlights the interde-pendence of epithelial, neural and endothelial cells; all of which generate NGF in normalgastric mucosa and interact during GU healing.
S-338AGA Abstracts
Sa1934
Importance of Thromboxane A2/Prostacyclin Balance in PathogenicMechanism of Ischemia/ Reperfusion-Induced Gastric Injury in MiceKoji Takeuchi, Yuka Nakamori, Shinji Kojima, Tohru Kotani
Background/Aim: Eicosanoids are involved in modulating the gastric mucosal integrity undervarious conditions. We previously reported that cyclooxygenase (COX)-2/ prostacyclin(PGI2) play a crucial role in the gastric mucosal defense under ischemia/ reperfusion (I/R)-induced conditions, whereas indomethacin worsens these lesions, at least partly, throughthe up-regulation of cysteinyl leukotrienes (CysLTs) and their receptors. In the presentstudy, we examined the role of thromboxane A2 (TXA2) in the ulcerogenic response in themouse stomach under I/R-induced conditions. Methods: Male C57BL/6J mice were usedafter 18 h fasting. Under urethane anesthesia, the celiac artery was clamped (ischemia) for30 min, followed by reperfusion by removal of the clamp. After reperfusion for 60 min, thearea of macroscopically visible lesions in the stomach was measured. Indomethacin (5 mg/kg) was administered SC 30 min before the onset of ischemia. SC-560 (COX-1 inhibitor:5 mg/kg), rofecoxib (COX-2 inhibitor: 5 mg/kg), ozagrel (TXA2 synthase inhibitor: 200mg/kg), seratrodast (TXA2 antagonist: 1-30 mg/kg) or pranlukast (CysLT receptor type 1antagonist: 10 mg/kg) was administered PO 60 min before ischemia. Iloprost (1 μg/kg) wasgiven IV 5 min before reperfusion. TXB2 and 6-keto-PGF1 α contents were measured byenzyme immunoassay. The gene expressions of TP receptor and TXA2 synthase were exam-ined by RT-PCR. Results: I/R produced hemorrhagic lesions in the gastric mucosa, and theseverity of these lesions was reduced by pretreatment with ozagrel, seratrodast or iloprost,in addition to pranlukast. By contrast, the gastric ulcerogenic response under I/R conditionswas worsened by pretreatment with indomethacin and rofecoxib but not SC-560, and thisaggravation was both abrogated by co-administration of iloprost. Pretreatment of ozagrel orseratrodast attenuated the worsening effect of rofecoxib but not the effect of indomethacinthat was abrogated by pranlukast. I/R significantly increased both TXB2 and 6-keto-PGF1acontents in the gastric mucosa. The increase of TXB2 content under I/R conditions wasattenuated by SC-560, while the increased PGI2 biosynthetic response was totally attenuatedby rofecoxib. I/R by itself had no effect on the TXB2/ 6-keto-PGF1a ratio in the gastricmucosa, yet the ratio was markedly increased by rofecoxib and decreased by ozagrel,respectively. The gene expressions of TP receptor and TXA2 synthase were observed in thenormal stomach and remained unchanged under I/R-induced conditions. Conclusion: Theseresults suggest that 1) COX-1/TXA2 plays a crucial role in the development of I/R-inducedgastric lesions, in addition to COX-2/PGI2 as well as CysLTs, and 2) the aggravation byrofecoxib of I/R-induced gastric lesions may be accounted partly for by the imbalance ofTXA2/PGI2 ratio in the stomach.
Sa1935
The Effect of Diabetes Mellitus on Lower Esophageal Sphincter Muscle andEsophageal Epithelia in RabbitDoga Capanoglu, Deniz Coskunsever, Murat Olukman, Serhat Bor
Gastrointestinal motility disorders were commonly seen in patients with Diabetes mellitus(DM). A weak relationship was reported between DM-GERD and with esophageal motilitydisturbances. The possible mechanisms are not known except neuropathy. We aimed toinvestigate the electrophysiological changes of diabetic rabbit esophageal epithelia (EE) andlower esophageal sphincter (LES) muscle using Ussing chamber system and isolated organbath. METHOD: Chronic diabetes in New Zealand white rabbits were created using alloxane(100mg/kg). Blood sugar levels were measured 72 hours after drug administration, valuesover 250mg/dl was accepted as diabetic. Rabbits were sacrificed at 8th week; EE was placedon Ussing chambers. Potential difference (PD), short circuit current (Isc) were measured,electrical resistance (R) was calculated. Acid-pepsin model was employed by titrating theringer solution of luminal side of the tissues to acidic (pH 1.7) or weakly acidic (pH 4)and/or addition of pepsin (0.25mg/ml). Tissues were fixed for morphological evaluation.Smooth muscle strips taken from LES were mounted to isolated organ bath, and kept inresting tension for 1 hour. Electrical Field Stimulation (EFS) and acetyl choline cumulativemuscle contraction response were recorded. These muscle contraction responses were alsotested in the presence of Rho-kinase inhibitor (Y-27632) and nonspecific nitric oxide inhibitor(L-NAME). RESULTS: The PD of diabetic tissues perfused in acidified ringer+pepsin dropped15%, while control tissues dropped 40%. Diabetic tissue resistance perfused in acidifiedringer solution decreased 10%, pepsin addition decreased 47%. Same concentrations causeda more distinct loss of resistance in control tissues (26% and 78%) (p ,0.05 compared tocontrol group). EFS responses was found to increase in diabetic group compared to control(p,0,001). Y-27632 and L-NAME was found to increase contraction response with higherconcentration without any statistical significance. The response increased with acetyl cholinein diabetic group with cumulative concentrations (p,0,001). In diabetic group; increasedconcentrations of Y-27632 was found to significantly decreased acetyl choline responses.This effect was not observed in control group. Histopathologic scores were the same betweengroups. DISCUSSION: Even though acid-pepsin model causes mucosal damage, diabeticrabbit esophageal tissues had less injury compared to control. The isolated organ bath studiessupported these findings; in diabetic rabbits, LES contraction response increased significantly,and Rho-Rhokinase pathway is found to be responsible of this change. These results canbe interpreted that, in chronic diabetic rabbit model, esophagus was found to resist againstreflux by mucosal defence mechanisms and increased contraction pressures.
Sa1936
Unique Protective Effect of Egualen, a Stable Azulene Derivative, onGastrointestinal Lesions Induced in Rats by Ischemia/Reperfusion, DoubleAntithrombotic Therapy, and LoxoprofenKikuko Amagase, Yuji Yoshida, Daisuke Hara, Toshiko Murakami, Koji Takeuchi
Background/Aim: Egualen Na (3-ethyl-7-isopropyl-1-azulemesulfonate 1/3 hydrate Na), astable azulene derivative, has been used as a mucosal protective drug for the treatment ofgastritis and gastric ulcers in Japan. This agent exhibits antiulcer activity against a variety
