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W1343 PDE 3 Inhibitor, Cilostazol, Ameriolates Small Intestinal Lesions Induced by Indomethacin
reactivation and chronicity of IBD. While previous In Vitro studies demonstrate the antiinflammatory capabilities of Prunella vulgaris and Hypericum gentianoides extracts, this potential has never been tested in an In Vivo model of colitis. In this context, we examined the ability of P. vulgaris or H. gentianoides oral botanical extracts to prevent or attenuate spontaneous typhlocolitis in mdr1a-/- mice. P. vulgaris (2.4 mg/day) or H. gentianoides (4.8 mg/day) were prepared by soxhlet extraction in 95% ethanol and were administered orally to mdr1a-/mice. Metronidazole, previously shown to prevent mdr1a-/- colitis via partial depletion of the intestinal microbiota, was included as a negative control. Differences in disease onset and severity were assessed by changes in colon length, typhlocolitic macroscopic score, microscopic histologic inflammatory score, and post-mortum serum cytokine and chemokine profiles. Administration of either P. vulgaris or H. gentianoides delayed clinical onset of colitis, increased colon lengths and reduced typhlocolitic scores in mdr1a-/- mice. Four of 6 shamtreated mice presenting with severe clinical signs of colitis were removed prior to the completion of the study due to excessive weight loss, while only 1 of 6 Prunella-treated and 2 of 6 Hypericum-treated mice were prematurely removed. Compared to sham-treated mice, serum levels of IL-10, TNFα, KC, IP-10, MIG and GM-CSF were significantly (p<0.05) reduced in P. vulgaris-treated mdr1a-/- mice, while IL-10, TNFα, IL-12(p70), KC, IP-10, MIG and LIX were significantly (p<0.05) decreased in mice treated with H. gentianoides. Our findings indicate that daily oral treatment with P. vulgaris or H. gentianoides extracts was associated with delayed onset and reduced magnitude of colitis in mdr1a-/- mice. Moreover, extract treatments were positively correlated with decreased severity of macroscopic and microscopic histologic inflammatory indices. Mice also exhibited decreased serum concentrations of pro-inflammatory cytokines and chemokines which drive mucosal inflammation and inflammatory immune cell recruitment. These studies indicate a beneficial role for orally administered Prunella or Hypericum as novel nutraceutical therapies as alternatives for biologic or steroidal parenteral therapies (e.g., infliximab or 5-aminosalicylates) for treating the chronic intestinal inflammation characteristic of IBD.
A Novel Approach for Gene Therapy in IBD Hamed Laroui, Guillaume Dalmasso, Hang Thi Thu Nguyen, Yutao Yan, Shanthi V. Sitaraman, Didier Merlin Background and aims: Standard drug delivery systems for IBD release anti-inflammatory drugs non-specifically to both healthy and inflamed colonic epithelium. However, since the use of drugs such as TNFα inhibitors has been associated with an increased risk of side effects related to immunosuppression, it would be highly beneficial to target controlled delivery of TNFα inhibitors to disease sites. Thus, we herein seek to investigate the effect of TNFα siRNA loaded nanoparticles (NP) in inflamed cells. Methods: Chitosan (CS) is a polysaccharide and has been widely used especially in gene delivery systems, due to its positively charged amines allowing electrostatic interactions with nucleic acids to form stable complexes. The complexation of TNFα siRNA with CS was performed to form the positively charged polyplex (TNFα siRNA/CS). Then TNFα siRNA/CS was loaded into polyvinylic alcohol (PVA)-covered D,L PLA NP as described previously (Gastroenterology, In Press). Results: The size distribution of TNFα siRNA/CS loaded NP revealed the average particle size evaluated by photon correlation spectroscopy (PCS) to be about 320 nm, whereas that evaluated by SEM was about 300 nm. We demonstrated that siRNA/CS loaded NP at a concentration of 1 mg/mL do not cause cellular death of epithelial cells (Caco2-BBE, IEC6) and immune cells (J774, RAW 264.7) over a 72h period compared with controls. Because TNFα is mainly produced by immune cells, we have investigated TNFα siRNA/CS uptake by Raw 264.7 a mice macrophage cell line well known to over expresses TNFα in response to lipopolysaccharide (LPS) stimulation. First using a fluorescent tagged TNFα siRNA/CS, we observed that FITC TNFα siRNA/CS were highly taken up into cells whereas FITC TNFα siRNA non complexed was not. Stimulated LPS Raw 264.7 cells were incubated with free TNFα siRNA/CS alone at concentration 30nM or with 30nM TNFα siRNA/CS loaded NP at a suspension concentration of 500μg/mL for 48 hours. We found that free TNFα siRNA/ CS was not taken up by Raw 264.7 cells and so the increased in LPS-induced TNFα expression levels was not reduced. In contrast TNFα siRNA/CS-loaded NP were clearly taken up by Raw 264.7 and that the increased in LPS-induced TNFα expression levels was reduced by 85%. Conclusion: Of the particle-based drug carriers, nanotechnology has recently shown great promises for promoting drug efficiency. Here, we demonstrate that TNFα siRNA may be effectively delivered to immune cells. The biodegradable polymeric envelope, that provides protection and transport of the siRNA into the cytosol and allow the siRNA to be efficient In Vivo. We are currently applying this strategy by using In Vivo approaches.
W1343 PDE 3 Inhibitor, Cilostazol, Ameriolates Small Intestinal Lesions Induced by Indomethacin Masaaki Higashiyama, Ryota Hokari, Chie Kurihara, Hisayuki Matsunaga, Toshihide Ueda, Chikako Watanabe, Yoshikiyo Okada, Kanji Wakabayashi, Mitsuyasu Nakamura, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are known to cause ulcers in the small intestine frequently. Ulcer inducing mechanism is more complicated than that of stomach and effective drugs are being in search for. Recruitment of neutrophils and monocytes is thought to be one of major factors to predispose intestinal lesions. Recently, we reported platelets play an important role for monocyte migration in inflamed small intestine and cilostazol, a phosphodiesterase (PDE) 3 inihibitor, attenuates monocyte migration via suppression of interaction between platelets and monocytes (Microcirculation 2008) and ameliorates chronic ileitis by decreasing monocyte recruitment (Am J Physiol 2009). The aim of this study was to clarify the therapeutic effectiveness of cilostazol on small intestinal lesions induced by indomethacin (IND). Subjects and methods: IND (2.5mg/kg) was peritoneally injected to C57/B6 daily for 4days. Cilostazol (100mg/kg) or anti PSGL-1 (2mg/kg) antibody was injected peritoneally one hour before the injection of IND in some mice. On day 5, the number of intestinal lesions stained dark blue with evans blue was counted. Myeloperoxidase (MPO) activity was examined by coloriometric assay procedure. Degree of mRNA expression of TNFα, IFNγ, intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM) and monocyte chemoattractant protein (MCP)-1 was examined by RT-PCR. Intestinal permeability was examined by concentration of dextran-FITC in urine 3 hours after 40mg of oral dextran-FITC administration. Monocytes from bone marrow and platelets from peripheral blood were labeled with CFSE in ex vivo and the change of their migration was evaluated under intravital microscopy in each group. Results: IND induced small intestinal damage with an increase of MPO activity, TNF-α, ICAM-1 and MCP-1 mRNA expressions. Intravital micoroscopy revealed monocytes adhesion as well as platelets to intestinal microvessels increased by IND treatment. Treatment with cilostazol or anti-PSGL-1 antibody not only attenuated monocytes recruitment but ameriolated small intestinal lesions with decrease in MPO activity and TNF-α. Intestinal permeability was also ameliorated. Conclusion: Recruitment of monocytes to intestinal microvessels turned out to be one of major factors to predispose intestinal lesions. Monocytes migration was blocked not only by anti-adhesion antibody treatment but by anti- platelet drug possibly by blocking interaction between monocytes and platelets. We propose cilostazol as possible promising therapy for NSAIDs induced intestinal injury.
W1341 Endogenous Prostaglandin E2 Accelerates Healing of Indomethacin-Induced Small Intestinal Lesions Through up-Regulation of VEGF via Activation of EP4 Receptors Kikuko Amagase, Azusa Kojo, Akimu Ochi, Koji Takeuchi Background/Aim: Endogenous prostaglandins (PGs)/cyclooxygenase (COX) play an important role in the healing of lesions in the small intestine as well as the stomach. We recently reported that both COX-1 (the late phase) and COX-2 (the early phase) are involved in the healing of indomethacin-induced small intestinal lesions, yet the mechanism how PGs promote the healing of these lesions remains unknown. In the present study, we investigated the mechanism by which PGs promote the healing of intestinal lesions in rats, including the determination of EP receptor subtype(s) involved in this action and the relation with VEGF expression. Methods: Male SD rats were used without fasting. Intestinal lesions were induced by a single injection of indomethacin (10 mg/kg, SC), and the animals were killed 1, 2, 3, 4, 5 and 7 days later. Indomethacin (2 mg/kg) was given PO once daily for 6 days, starting 1 day after ulceration. In some cases, AE1-329 (EP4 agonist: 0.1 mg/kg) was given IP once daily for 6 days in the presence of indomethacin. For studying VEGF expression, indomethacin (2 mg/kg) and AE3-208 (EP4 antagonist: 3 mg/kg) were given PO once daily for 2 days, while AE1-329 (EP4 agonist: 10 μg/kg) was given PO twice daily together with indomethacin (2 mg/kg, PO). PGE2 content was measured by EIA. VEGF expression in the intestinal mucosa was examined by western blotting. Results: Indomethacin (10 mg/kg) caused severe lesions in the small intestine, and the lesions healed quite rapidly within 7 days, the area of lesions becoming less than 1/10 of the initial area. The healing of these lesions was significantly impaired by the repeated treatment of indomethacin (2 mg/kg) given once daily for 6 days. The healing impairment effect of indomethacin was mimicked by the EP4 antagonist AE3-208 given for 6 days and significantly reversed by the coadministration of 16,16-dimethyl PGE2 as well as the EP4 agonist AE1-329. The COX-2 expression in the small intestine was up-regulated after ulceration, persisting for 3 days, while the muosal VEGF expression was increased from 1 day after ulceration, reaching a peak on day 3 followed by a decrease and the changes in VEGF expression were in good parallel with those in mucosal PGE2 content. Indomethacin (2 mg/kg) down-regulated both the expression of VEGF and the number of microvessels in the mucosa, and these effects were significantly reversed by the co-administration with AE1-329. Conclusion: These results suggest that endogenous PGE2 plays an important role in the healing of small intestinal lesions, through the stimulation of angiogenesis via the up-regulation of VEGF expression mediated by the activation of EP4 receptors.
W1344 Intestinal Directed Adeno-Associated Virus Expressing IL-10 Improves Enterocolitis in a Murine Model of Inflammatory Bowel Disease Steven Polyak, Stacy L. Porvasnik, Thomas Conlon, Annette Mach, Clive Wasserfall, Lisa R. Dixon, Cathryn Mah Background: Inflammatory bowel disease is a chronic relapsing illness that requires lifelong therapy. Our best treatments to date offer only 40% long term response rates and many of these medications have significant side effects. The need for more efficacious and localized therapy is clear. Prior work using systemic gene transfer approaches in an IL-10 knockout mouse model of colitis, demonstrate only minimal improvement. Selective intestinal transduction for localized expression of mucosal IL-10, could offer a more effective approach to modulate intestinal inflammation and improve chronic colitis. Our aim was to determine if AAV mediated intestinal expression of IL-10 transgene could clinically improve the IL-10 knockout model of enterocolitis. Methods: 5-6 wk old male IL-10 knockout mice (C57Bl6) were treated with 1x10^11 vg of AAV10-IL10 and AAV10-GFP via superior mesenteric artery (SMA) and portal vein (PV) injections (n=5). Enterocolitis was initiated and synchronized with Piroxicam. Clinical data (weight, stool consistency and blood in stool) and serum were serially collected. Mice were sacrificed 30 days after onset of enterocolitis (60 days post AAV treatment) for histopathologic analysis. Results: Serum levels of IL-10 were maintained throughout the experiment in SMA injected mice. PV injected animals had an early increase
W1342 Prunella Vulgaris and Hypericum Gentianoides Oral Extracts Attenuate Spontaneous Typhlocolitis in Mdr1a Deficient (-/-) Mice Kelley M. Haarberg, Catherine Hauck, Patricia A. Murphy, Jia Liu, Philip Dixon, Jesse Hostetter, Michael J. Wannemuehler Inflammatory bowel disease (IBD) involves disruption of gut mucosal homeostasis, loss of epithelial barrier integrity and immunologic response to the gut microbiota, skewing the gut milieu towards chronic inflammation. These perturbations likely contribute to the onset,
S-703
AGA Abstracts
AGA Abstracts
W1340
A Novel Approach for Gene Therapy in IBD Hamed Laroui, Guillaume Dalmasso, Hang Thi Thu Nguyen, Yutao Yan, Shanthi V. Sitaraman, Didier Merlin Background and aims: Standard drug delivery systems for IBD release anti-inflammatory drugs non-specifically to both healthy and inflamed colonic epithelium. However, since the use of drugs such as TNFα inhibitors has been associated with an increased risk of side effects related to immunosuppression, it would be highly beneficial to target controlled delivery of TNFα inhibitors to disease sites. Thus, we herein seek to investigate the effect of TNFα siRNA loaded nanoparticles (NP) in inflamed cells. Methods: Chitosan (CS) is a polysaccharide and has been widely used especially in gene delivery systems, due to its positively charged amines allowing electrostatic interactions with nucleic acids to form stable complexes. The complexation of TNFα siRNA with CS was performed to form the positively charged polyplex (TNFα siRNA/CS). Then TNFα siRNA/CS was loaded into polyvinylic alcohol (PVA)-covered D,L PLA NP as described previously (Gastroenterology, In Press). Results: The size distribution of TNFα siRNA/CS loaded NP revealed the average particle size evaluated by photon correlation spectroscopy (PCS) to be about 320 nm, whereas that evaluated by SEM was about 300 nm. We demonstrated that siRNA/CS loaded NP at a concentration of 1 mg/mL do not cause cellular death of epithelial cells (Caco2-BBE, IEC6) and immune cells (J774, RAW 264.7) over a 72h period compared with controls. Because TNFα is mainly produced by immune cells, we have investigated TNFα siRNA/CS uptake by Raw 264.7 a mice macrophage cell line well known to over expresses TNFα in response to lipopolysaccharide (LPS) stimulation. First using a fluorescent tagged TNFα siRNA/CS, we observed that FITC TNFα siRNA/CS were highly taken up into cells whereas FITC TNFα siRNA non complexed was not. Stimulated LPS Raw 264.7 cells were incubated with free TNFα siRNA/CS alone at concentration 30nM or with 30nM TNFα siRNA/CS loaded NP at a suspension concentration of 500μg/mL for 48 hours. We found that free TNFα siRNA/ CS was not taken up by Raw 264.7 cells and so the increased in LPS-induced TNFα expression levels was not reduced. In contrast TNFα siRNA/CS-loaded NP were clearly taken up by Raw 264.7 and that the increased in LPS-induced TNFα expression levels was reduced by 85%. Conclusion: Of the particle-based drug carriers, nanotechnology has recently shown great promises for promoting drug efficiency. Here, we demonstrate that TNFα siRNA may be effectively delivered to immune cells. The biodegradable polymeric envelope, that provides protection and transport of the siRNA into the cytosol and allow the siRNA to be efficient In Vivo. We are currently applying this strategy by using In Vivo approaches.
W1343 PDE 3 Inhibitor, Cilostazol, Ameriolates Small Intestinal Lesions Induced by Indomethacin Masaaki Higashiyama, Ryota Hokari, Chie Kurihara, Hisayuki Matsunaga, Toshihide Ueda, Chikako Watanabe, Yoshikiyo Okada, Kanji Wakabayashi, Mitsuyasu Nakamura, Atsushi Kawaguchi, Shigeaki Nagao, Soichiro Miura Abstract: Non-steroidal anti-inflammatory drugs (NSAIDs) are known to cause ulcers in the small intestine frequently. Ulcer inducing mechanism is more complicated than that of stomach and effective drugs are being in search for. Recruitment of neutrophils and monocytes is thought to be one of major factors to predispose intestinal lesions. Recently, we reported platelets play an important role for monocyte migration in inflamed small intestine and cilostazol, a phosphodiesterase (PDE) 3 inihibitor, attenuates monocyte migration via suppression of interaction between platelets and monocytes (Microcirculation 2008) and ameliorates chronic ileitis by decreasing monocyte recruitment (Am J Physiol 2009). The aim of this study was to clarify the therapeutic effectiveness of cilostazol on small intestinal lesions induced by indomethacin (IND). Subjects and methods: IND (2.5mg/kg) was peritoneally injected to C57/B6 daily for 4days. Cilostazol (100mg/kg) or anti PSGL-1 (2mg/kg) antibody was injected peritoneally one hour before the injection of IND in some mice. On day 5, the number of intestinal lesions stained dark blue with evans blue was counted. Myeloperoxidase (MPO) activity was examined by coloriometric assay procedure. Degree of mRNA expression of TNFα, IFNγ, intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM) and monocyte chemoattractant protein (MCP)-1 was examined by RT-PCR. Intestinal permeability was examined by concentration of dextran-FITC in urine 3 hours after 40mg of oral dextran-FITC administration. Monocytes from bone marrow and platelets from peripheral blood were labeled with CFSE in ex vivo and the change of their migration was evaluated under intravital microscopy in each group. Results: IND induced small intestinal damage with an increase of MPO activity, TNF-α, ICAM-1 and MCP-1 mRNA expressions. Intravital micoroscopy revealed monocytes adhesion as well as platelets to intestinal microvessels increased by IND treatment. Treatment with cilostazol or anti-PSGL-1 antibody not only attenuated monocytes recruitment but ameriolated small intestinal lesions with decrease in MPO activity and TNF-α. Intestinal permeability was also ameliorated. Conclusion: Recruitment of monocytes to intestinal microvessels turned out to be one of major factors to predispose intestinal lesions. Monocytes migration was blocked not only by anti-adhesion antibody treatment but by anti- platelet drug possibly by blocking interaction between monocytes and platelets. We propose cilostazol as possible promising therapy for NSAIDs induced intestinal injury.
W1341 Endogenous Prostaglandin E2 Accelerates Healing of Indomethacin-Induced Small Intestinal Lesions Through up-Regulation of VEGF via Activation of EP4 Receptors Kikuko Amagase, Azusa Kojo, Akimu Ochi, Koji Takeuchi Background/Aim: Endogenous prostaglandins (PGs)/cyclooxygenase (COX) play an important role in the healing of lesions in the small intestine as well as the stomach. We recently reported that both COX-1 (the late phase) and COX-2 (the early phase) are involved in the healing of indomethacin-induced small intestinal lesions, yet the mechanism how PGs promote the healing of these lesions remains unknown. In the present study, we investigated the mechanism by which PGs promote the healing of intestinal lesions in rats, including the determination of EP receptor subtype(s) involved in this action and the relation with VEGF expression. Methods: Male SD rats were used without fasting. Intestinal lesions were induced by a single injection of indomethacin (10 mg/kg, SC), and the animals were killed 1, 2, 3, 4, 5 and 7 days later. Indomethacin (2 mg/kg) was given PO once daily for 6 days, starting 1 day after ulceration. In some cases, AE1-329 (EP4 agonist: 0.1 mg/kg) was given IP once daily for 6 days in the presence of indomethacin. For studying VEGF expression, indomethacin (2 mg/kg) and AE3-208 (EP4 antagonist: 3 mg/kg) were given PO once daily for 2 days, while AE1-329 (EP4 agonist: 10 μg/kg) was given PO twice daily together with indomethacin (2 mg/kg, PO). PGE2 content was measured by EIA. VEGF expression in the intestinal mucosa was examined by western blotting. Results: Indomethacin (10 mg/kg) caused severe lesions in the small intestine, and the lesions healed quite rapidly within 7 days, the area of lesions becoming less than 1/10 of the initial area. The healing of these lesions was significantly impaired by the repeated treatment of indomethacin (2 mg/kg) given once daily for 6 days. The healing impairment effect of indomethacin was mimicked by the EP4 antagonist AE3-208 given for 6 days and significantly reversed by the coadministration of 16,16-dimethyl PGE2 as well as the EP4 agonist AE1-329. The COX-2 expression in the small intestine was up-regulated after ulceration, persisting for 3 days, while the muosal VEGF expression was increased from 1 day after ulceration, reaching a peak on day 3 followed by a decrease and the changes in VEGF expression were in good parallel with those in mucosal PGE2 content. Indomethacin (2 mg/kg) down-regulated both the expression of VEGF and the number of microvessels in the mucosa, and these effects were significantly reversed by the co-administration with AE1-329. Conclusion: These results suggest that endogenous PGE2 plays an important role in the healing of small intestinal lesions, through the stimulation of angiogenesis via the up-regulation of VEGF expression mediated by the activation of EP4 receptors.
W1344 Intestinal Directed Adeno-Associated Virus Expressing IL-10 Improves Enterocolitis in a Murine Model of Inflammatory Bowel Disease Steven Polyak, Stacy L. Porvasnik, Thomas Conlon, Annette Mach, Clive Wasserfall, Lisa R. Dixon, Cathryn Mah Background: Inflammatory bowel disease is a chronic relapsing illness that requires lifelong therapy. Our best treatments to date offer only 40% long term response rates and many of these medications have significant side effects. The need for more efficacious and localized therapy is clear. Prior work using systemic gene transfer approaches in an IL-10 knockout mouse model of colitis, demonstrate only minimal improvement. Selective intestinal transduction for localized expression of mucosal IL-10, could offer a more effective approach to modulate intestinal inflammation and improve chronic colitis. Our aim was to determine if AAV mediated intestinal expression of IL-10 transgene could clinically improve the IL-10 knockout model of enterocolitis. Methods: 5-6 wk old male IL-10 knockout mice (C57Bl6) were treated with 1x10^11 vg of AAV10-IL10 and AAV10-GFP via superior mesenteric artery (SMA) and portal vein (PV) injections (n=5). Enterocolitis was initiated and synchronized with Piroxicam. Clinical data (weight, stool consistency and blood in stool) and serum were serially collected. Mice were sacrificed 30 days after onset of enterocolitis (60 days post AAV treatment) for histopathologic analysis. Results: Serum levels of IL-10 were maintained throughout the experiment in SMA injected mice. PV injected animals had an early increase
W1342 Prunella Vulgaris and Hypericum Gentianoides Oral Extracts Attenuate Spontaneous Typhlocolitis in Mdr1a Deficient (-/-) Mice Kelley M. Haarberg, Catherine Hauck, Patricia A. Murphy, Jia Liu, Philip Dixon, Jesse Hostetter, Michael J. Wannemuehler Inflammatory bowel disease (IBD) involves disruption of gut mucosal homeostasis, loss of epithelial barrier integrity and immunologic response to the gut microbiota, skewing the gut milieu towards chronic inflammation. These perturbations likely contribute to the onset,
S-703
AGA Abstracts
AGA Abstracts
W1340