Mo2069 Effect of Intestinal Extracelluar Matrix on Endothelial Biology and Angiogenesis
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of TSA reduced the arginase I expression four fold in M2a cells, while IL-6 transcript in M2a was four fold increased. Interleukin-6 transcript level in inflammatory M1 cells was reduced two fold after polarization with IFN-γ and TSA. In M2a cells expressed significantly lower HDAC 7 levels compared to M1 cells (p<0.05) after 3 days of polarization. Human M1, M2a and M2c macrophages expressed the polarization markers CD64, CD200R and CD163 respectively. No significant differences were found in HDAC 1-11 and sirtuin 1 expression levels between MDM subsets directly after polarization. LPS stimulation of MDM in the presence of TSA showed that HDAC inhibition had no effect on TNF-α or IL-6 secretion, but IL-10 secretion was abrogated almost completely in all polarized macrophage MDM subsets. CONCLUSION HDAC activity is involved in macrophage polarization process. Inhibition of HDAC activity changes cytokine responses of macrophage M2a to inflammatory stimuli. Together, these data underscore the potential of HDAC inhibition to intervene in macrophage differentiation and reactivity in inflammatory disease. Mo2069 Effect of Intestinal Extracelluar Matrix on Endothelial Biology and Angiogenesis William M. Rivers, Annette Wilson, Arthur Barrie, Anthony J. Bauer, David G. Binion Background: The extracellular matrix (ECM) plays a critical role in cell attachment, prolifera- tion, migration and angiogenesis. Tissue regeneration protocols emphasize the use of organ specific ECM to facilitate cell reconstitution, where revascularization is an early event. We generated human and mouse specific intestinal ECM and characterized its effects on the In Vitro growth of human intestinal microvascular endothelial cells (HIMEC) as well as its In Vivo capacity for revascularization when implanted into the subcutaneous tissues in mice. Methods: ECM was decellularized and isolated from full thickness sections of human and mouse intestine. Complete decellularization of the bowel and the preservation of the vascular network were confirmed. Scanning electron microscopy (SEM) showed preserved structure of decellularized tissue. HIMECs were cultured on plates coated with either normal or diseased ECM or fibronectin and assessed morphologically. Low-flow shear-stress leukocyte adhesion with RPMI 8866 cells expressing the alpha-4 beta-7 integrin which selectively binds MAdCAM-1 expressed on HIMECs, was also performed. ECM from colons of mice undergoing treatment with dextran sulfate sodium to induce colitis were implanted into a subcutaneous pocket in the backs of C57Bl/6 mice. The ECM was retrieved after 3 weeks, 2 months, and 3 months. Results: The HIMECs grown on both normal and diseased ECM had normal endothelial characteristics and morphology. Cells displayed the classic morphology and dil-Ac-LDL uptake similar to the HIMECs grown on fibronectin. The HIMECs also stained positive for PECAM-1. All 3 groups of HIMEC cells - fibronectin- coated, normal ECM-coated, and diseased ECM-coated displayed similar adhesion properties under varying conditions: Control-normal ECM and Control-diseased ECM showed no change in adhesion from the Control-fibronectin. All groups of HIMECs activated with TNF- a/LPS showed the same fold-increase from the Control (Fibronectin-coated = 14.75, Normal ECM-coated = 15.01, Diseased ECM-coated = 15.05). Recovery of ECM at 3 weeks and 2 months showed re-growth of blood vessels in the ECM as shown in whole mounts. In addition, the ECM recovered at both time points showed substantial growth of fibrotic masses, muscularis propria, and epithelium as shown in H&E stained sections. Conclusions: The ECM isolated from the human intestine successfully supported the normal growth of primary HIMECs. In addition, the function of the cells grown on both normal and diseased ECM functioned normally in adhesion studies using RPMI 8866 cell line. The In Vitro studies of ECM isolated from mouse colon implanted in a subcutaneous pouch in a mouse model showed that the ECM became vascularized and histology indicated cellular growth on the ECM. Mo2070 Modulatory Effect of Hypoxia on Inflammatory and Angiogenic Activation of Human Intestinal Microvascular Endothelial Cells Annette Wilson, William M. Rivers, Anthony J. Bauer, Arthur Barrie, David G. Binion Background. Mild cellular stress due to hypoxia will induce homeostatic mechanisms which can modulate subsequent activation due to potent inflammatory or angiogenic stimuli. Hypoxia-inducible factor (HIF) signaling is known to protect the intestinal mucosa from insults by toxins. HIF-1a increases under hypoxic conditions and may by induced by treatment with the chemical Dimethyloxaloylglycine (DMOG). The purpose of this study to determine if treatment of primary human microvascular endothelial cells (HIMEC) with hypoxia or DMOG will have an effect on the cell growth and adhesion response to TNF-a/ LPS. Methods: Cell growth of HIMECs was observed under normoxia (21% O2) and hypoxia (2% O2) conditions for exposure times of 1, 3, 5, and 7 days. In a separate experiment, HIMECs that were undergoing endothelial mesenchymal transition (EndMT) were put into normoxia and hypoxia conditions immediately upon EndMT for exposure times of up to 10 days. Finally, low-flow shear-stress leukocyte adhesion with RPMI 8866 cells, human B lymphoma cell line known to specifically express the integrin alpha-4 beta-7 that bind selectively in the gut to MAdCAM-1 expressed on the surface of HIMECs, was performed under normoxia, hypoxia, and DMOG conditions to study the effect on activation with TNF-a/LPS. Results: The late passage HIMECs showed changes in morphology under the normoxia conditions by day 3 while the morphology of the cells in hypoxia conditions remained normal. Phalloidin staining of the cells undergoing morphologic change showed thickening of F-actin stress fibers. Hypoxia conditions did not have any effect on the cells that had undergone EndMT. The low-flow shear-stress leukocyte adhesion studies using RPMI 8866 showed that hypoxia and DMOG treatments to increase HIF-1a appear to attenuate the activation of HIMECs by TNF-a/LPS treatment as shown by lowered RPMI 8866 adhesion to HIMECs. The optimum concentration of 0.5mM DMOG significantly attenuated the adhesion response from TNF-a/LPS activation in comparison to HIMECs that did not receive DMOG treatment (5 fold increase from unactivated HIMECs vs 20 fold increase from unactivated HIMECs). HIMECs treated with 2% hypoxia for 48 H followed by TNF/LPS activation resulted in a fold increase from control of 6 while cells under normoxia condition showed a response of 15 fold increase from control. Conclusion: These findings suggest that hypoxic conditioning of HIMEC as well as agents known to induce HIF-1a S-733 AGA Abstracts may not only attenuate inflammatory activation and enhanced leukocyte adhesion but may also upregulate wound repair mechanisms including angiogenesis. Tu1056 Beclomethasone Dipropionate Topical Rectal Treatment for the Prevention of Rectal Bleeding in Patients Irradiated for Prostate Cancer: A Cost- Effectiveness Analysis Based on a Randomized Controlled Trial Lorenzo Fuccio, Cesare Hassan, Alessandra Guido, Liboria Laterza, Franco Bazzoli Background: Prostate cancer is the second most frequently diagnosed cancer worldwide and radiotherapy is an established treatment modality. Radiation-induced rectal bleeding is a frequently reported side effect, that impair patient's quality of life. A recently published randomized, double-blind, placebo-controlled trial showed that preventive treatment with topical rectal beclomethasone dipropionate (BDP) significantly reduces the risk of radiation- induced rectal bleeding and improves patient's quality of life (Fuccio L et al. Aliment Pharmacol Ther 2011). Aim of this study was to assess the cost-effectiveness of preventive treatment with topical rectal BDP in patients irradiated for prostate cancer. Patients and Methods: From June 2007 to October 2008, 120 patients were randomized to the BDP (n = 60) and placebo (n = 60) arms and were followed up for 12 months. Patients were treated with a 3 mg BDP enema or identical looking placebo for the entire duration of radiotherapy and, immediately after the end of radiotherapy, patients stopped the enema formulation and received two 3 mg BDP suppositories or identical placebo, for 4 more weeks. Quality of life was measured using the patient's quality of the modified Inflammatory Bowel Disease Quality of Life Index (IBDQ). Analysis was by intention-to-treat. Costs incurring in each arm were also assessed, including costs for BDP treatment, hyperbaric oxygen therapy, endoscopic treatment and primary care physicians visits. Incremental cost-effectiveness ratios were estimated as cost per quality-adjusted life year (QALY) gained. Results: After 12 months of follow-up the reduction of the total IBDQ scores was significantly more pronounced for patients on placebo (mean IBDQ±SE; BDP vs PL: 213,6±2.3 vs 203.9±4.0; p=0.034). The per patient difference in cost between the two arms (BDP vs placebo) was 80 euro. This was due to the net cost of the drug (164 euro per patient) in the BDP arm partially reduced by the additional cost for hyperbaric oxygen therapy in the placebo group (84 euro per patient), because of the higher incidence of severe bleeding in the placebo arm. The corres- ponding per patient difference in QALY at the end of a 12 month-follow up was 0.06 QALY in favour of the BDP arm. The corresponding cost-effectiveness ratio was equal to 1,330 euro per QALY gained, that is well below the threshold of 80,000 euro per QALY gained generally used to differentiate between cost-effective and -not effective interventions. Conclu- sions: Preventive treatment of radiation-induced rectal bleeding with topical rectal beclome- thasone dipropionate is a cost-effective preventive strategy in patients irradiated for pro- state cancer. Tu1057 Favorable Cost-Benefit Analysis Justifies the Implementation of Higher Sensitivity Molecular Testing for C. difficile Peter Dryer, Bruce R. Yacyshyn, Halim Muslu, Jesse Pratt, Shaun Chandna, Christina N. Naumovich Introduction: C. difficile is the most commonly recognized cause of infectious diarrhea in hospitalized patients. Enzyme immunoassay (EIA) testing has lower sensitivity relative to other methods, yet it has become commonplace due to cost. Sensitivity of ELISA testing is about 60-90%, while newer molecular assays are about 95%. Some have suggested utility in testing multiple samples by EIA to circumvent the lower sensitivities, especially in C. difficile associated diarrhea (CDAD) epidemics, but others have found limited data to support this. Repeated testing also delays diagnosis. The objective of this study was to determine the cost-benefit of testing inpatients with a less sensitive EIA-based test for C. difficile toxin versus a more sensitive molecular assay. Methods: A retrospective chart review was performed of inpatients diagnosed with CDAD by EIA at our institution during an 11 month period. Those with previous diagnosis of CDAD within 30 days were excluded. Patients were divided between those diagnosed with first EIA test versus multiple, and median hospital costs obtained. This analysis assumes that EIA testing more often relies on multiple testing, while molecular assays do not. Cost drivers were documented: need for consult, endoscopy, CT scan, abdominal ultrasound. A clinical assessment, per recent guidelines, of CDAD severity was made. Additionally, risk factors for infection were documented: length of stay prior to infection, age, antibiotic use within 14 days, previous CDAD more than 30 days prior, type of antibiotic for CDAD, immunosuppression (chemotherapy, immunosuppressive medication, ESRD, DM), tube feeding, acid blocking medication, inflammatory bowel disease, recent bowel preparation or surgery. The incremental cost difference between the groups was determined. Statistical differences in each category of clinical data were examined. A hypothet- ical annual savings was determined, assuming diagnosis on the first sample in all patients. Results: 156 patients met criteria. Of these, 101 were diagnosed after 1 EIA test, and 55 were diagnosed after multiple. The median incremental cost increase for those requiring multiple tests prior to diagnosis with CDAD was $48,356 per patient, or a total of about $2.66 million over this period. However, all cost drivers and nearly all clinical risk factors were equally distributed. Only DM was found to be significantly greater in the group requiring multiple EIA tests. Conclusion: This cost-benefit analysis supports the hypothesis that significant cost savings are achieved for inpatients diagnosed with CDAD with higher sensitivity testing techniques rather than EIA. Adjusted annually at our institution, these accrued annual savings are 2.9 million dollars. Use of newer molecular assays, with far superior sensitivity, may be able to realize most of the potential savings. AGA Abstracts
Transcript of Mo2069 Effect of Intestinal Extracelluar Matrix on Endothelial Biology and Angiogenesis
of TSA reduced the arginase I expression four fold in M2a cells, while IL-6 transcript inM2a was four fold increased. Interleukin-6 transcript level in inflammatory M1 cells wasreduced two fold after polarization with IFN-γ and TSA. In M2a cells expressed significantlylower HDAC 7 levels compared to M1 cells (p<0.05) after 3 days of polarization. HumanM1, M2a and M2c macrophages expressed the polarization markers CD64, CD200R andCD163 respectively. No significant differences were found in HDAC 1-11 and sirtuin 1expression levels between MDM subsets directly after polarization. LPS stimulation of MDMin the presence of TSA showed that HDAC inhibition had no effect on TNF-α or IL-6secretion, but IL-10 secretion was abrogated almost completely in all polarized macrophageMDM subsets. CONCLUSIONHDAC activity is involved inmacrophage polarization process.Inhibition of HDAC activity changes cytokine responses of macrophage M2a to inflammatorystimuli. Together, these data underscore the potential of HDAC inhibition to intervene inmacrophage differentiation and reactivity in inflammatory disease.
Mo2069
Effect of Intestinal Extracelluar Matrix on Endothelial Biology andAngiogenesisWilliam M. Rivers, Annette Wilson, Arthur Barrie, Anthony J. Bauer, David G. Binion
Background: The extracellular matrix (ECM) plays a critical role in cell attachment, prolifera-tion, migration and angiogenesis. Tissue regeneration protocols emphasize the use of organspecific ECM to facilitate cell reconstitution, where revascularization is an early event. Wegenerated human and mouse specific intestinal ECM and characterized its effects on the InVitro growth of human intestinal microvascular endothelial cells (HIMEC) as well as its InVivo capacity for revascularization when implanted into the subcutaneous tissues in mice.Methods: ECM was decellularized and isolated from full thickness sections of human andmouse intestine. Complete decellularization of the bowel and the preservation of the vascularnetwork were confirmed. Scanning electron microscopy (SEM) showed preserved structureof decellularized tissue. HIMECs were cultured on plates coated with either normal ordiseased ECM or fibronectin and assessed morphologically. Low-flow shear-stress leukocyteadhesion with RPMI 8866 cells expressing the alpha-4 beta-7 integrin which selectivelybinds MAdCAM-1 expressed on HIMECs, was also performed. ECM from colons of miceundergoing treatment with dextran sulfate sodium to induce colitis were implanted into asubcutaneous pocket in the backs of C57Bl/6 mice. The ECM was retrieved after 3 weeks,2 months, and 3 months. Results: The HIMECs grown on both normal and diseasedECM had normal endothelial characteristics and morphology. Cells displayed the classicmorphology and dil-Ac-LDL uptake similar to the HIMECs grown on fibronectin. TheHIMECs also stained positive for PECAM-1. All 3 groups of HIMEC cells - fibronectin-coated, normal ECM-coated, and diseased ECM-coated displayed similar adhesion propertiesunder varying conditions: Control-normal ECM and Control-diseased ECM showed nochange in adhesion from the Control-fibronectin. All groups of HIMECs activated with TNF-a/LPS showed the same fold-increase from the Control (Fibronectin-coated = 14.75, NormalECM-coated = 15.01, Diseased ECM-coated = 15.05). Recovery of ECM at 3 weeks and 2months showed re-growth of blood vessels in the ECM as shown in whole mounts. Inaddition, the ECM recovered at both time points showed substantial growth of fibroticmasses, muscularis propria, and epithelium as shown in H&E stained sections. Conclusions:The ECM isolated from the human intestine successfully supported the normal growth ofprimary HIMECs. In addition, the function of the cells grown on both normal and diseasedECM functioned normally in adhesion studies using RPMI 8866 cell line. The In Vitro studiesof ECM isolated from mouse colon implanted in a subcutaneous pouch in a mouse modelshowed that the ECM became vascularized and histology indicated cellular growth onthe ECM.
Mo2070
Modulatory Effect of Hypoxia on Inflammatory and Angiogenic Activation ofHuman Intestinal Microvascular Endothelial CellsAnnette Wilson, William M. Rivers, Anthony J. Bauer, Arthur Barrie, David G. Binion
Background. Mild cellular stress due to hypoxia will induce homeostatic mechanisms whichcan modulate subsequent activation due to potent inflammatory or angiogenic stimuli.Hypoxia-inducible factor (HIF) signaling is known to protect the intestinal mucosa frominsults by toxins. HIF-1a increases under hypoxic conditions and may by induced bytreatment with the chemical Dimethyloxaloylglycine (DMOG). The purpose of this study todetermine if treatment of primary human microvascular endothelial cells (HIMEC) withhypoxia or DMOG will have an effect on the cell growth and adhesion response to TNF-a/LPS. Methods: Cell growth of HIMECs was observed under normoxia (21% O2) and hypoxia(2% O2) conditions for exposure times of 1, 3, 5, and 7 days. In a separate experiment,HIMECs that were undergoing endothelial mesenchymal transition (EndMT) were put intonormoxia and hypoxia conditions immediately upon EndMT for exposure times of up to10 days. Finally, low-flow shear-stress leukocyte adhesion with RPMI 8866 cells, human Blymphoma cell line known to specifically express the integrin alpha-4 beta-7 that bindselectively in the gut to MAdCAM-1 expressed on the surface of HIMECs, was performedunder normoxia, hypoxia, and DMOG conditions to study the effect on activation withTNF-a/LPS. Results: The late passage HIMECs showed changes in morphology under thenormoxia conditions by day 3 while the morphology of the cells in hypoxia conditionsremained normal. Phalloidin staining of the cells undergoing morphologic change showedthickening of F-actin stress fibers. Hypoxia conditions did not have any effect on the cellsthat had undergone EndMT. The low-flow shear-stress leukocyte adhesion studies usingRPMI 8866 showed that hypoxia and DMOG treatments to increase HIF-1a appear toattenuate the activation of HIMECs by TNF-a/LPS treatment as shown by lowered RPMI8866 adhesion to HIMECs. The optimum concentration of 0.5mM DMOG significantlyattenuated the adhesion response from TNF-a/LPS activation in comparison to HIMECs thatdid not receive DMOG treatment (5 fold increase from unactivated HIMECs vs 20 foldincrease from unactivated HIMECs). HIMECs treated with 2% hypoxia for 48 H followedby TNF/LPS activation resulted in a fold increase from control of 6 while cells under normoxiacondition showed a response of 15 fold increase from control. Conclusion: These findingssuggest that hypoxic conditioning of HIMEC as well as agents known to induce HIF-1a
S-733 AGA Abstracts
may not only attenuate inflammatory activation and enhanced leukocyte adhesion but mayalso upregulate wound repair mechanisms including angiogenesis.
Tu1056
Beclomethasone Dipropionate Topical Rectal Treatment for the Prevention ofRectal Bleeding in Patients Irradiated for Prostate Cancer: A Cost-Effectiveness Analysis Based on a Randomized Controlled TrialLorenzo Fuccio, Cesare Hassan, Alessandra Guido, Liboria Laterza, Franco Bazzoli
Background: Prostate cancer is the second most frequently diagnosed cancer worldwide andradiotherapy is an established treatment modality. Radiation-induced rectal bleeding is afrequently reported side effect, that impair patient's quality of life. A recently publishedrandomized, double-blind, placebo-controlled trial showed that preventive treatment withtopical rectal beclomethasone dipropionate (BDP) significantly reduces the risk of radiation-induced rectal bleeding and improves patient's quality of life (Fuccio L et al. AlimentPharmacol Ther 2011). Aim of this study was to assess the cost-effectiveness of preventivetreatment with topical rectal BDP in patients irradiated for prostate cancer. Patients andMethods: From June 2007 to October 2008, 120 patients were randomized to the BDP (n =60) and placebo (n = 60) arms and were followed up for 12 months. Patients were treatedwith a 3 mg BDP enema or identical looking placebo for the entire duration of radiotherapyand, immediately after the end of radiotherapy, patients stopped the enema formulationand received two 3 mg BDP suppositories or identical placebo, for 4 more weeks. Qualityof life was measured using the patient's quality of the modified Inflammatory Bowel DiseaseQuality of Life Index (IBDQ). Analysis was by intention-to-treat. Costs incurring in eacharm were also assessed, including costs for BDP treatment, hyperbaric oxygen therapy,endoscopic treatment and primary care physicians visits. Incremental cost-effectiveness ratioswere estimated as cost per quality-adjusted life year (QALY) gained. Results: After 12 monthsof follow-up the reduction of the total IBDQ scores was significantly more pronounced forpatients on placebo (mean IBDQ±SE; BDP vs PL: 213,6±2.3 vs 203.9±4.0; p=0.034). Theper patient difference in cost between the two arms (BDP vs placebo) was 80 euro. Thiswas due to the net cost of the drug (164 euro per patient) in the BDP arm partially reducedby the additional cost for hyperbaric oxygen therapy in the placebo group (84 euro perpatient), because of the higher incidence of severe bleeding in the placebo arm. The corres-ponding per patient difference in QALY at the end of a 12 month-follow up was 0.06 QALYin favour of the BDP arm. The corresponding cost-effectiveness ratio was equal to 1,330euro per QALY gained, that is well below the threshold of 80,000 euro per QALY gainedgenerally used to differentiate between cost-effective and -not effective interventions. Conclu-sions: Preventive treatment of radiation-induced rectal bleeding with topical rectal beclome-thasone dipropionate is a cost-effective preventive strategy in patients irradiated for pro-state cancer.
Tu1057
Favorable Cost-Benefit Analysis Justifies the Implementation of HigherSensitivity Molecular Testing for C. difficilePeter Dryer, Bruce R. Yacyshyn, Halim Muslu, Jesse Pratt, Shaun Chandna, Christina N.Naumovich
Introduction: C. difficile is the most commonly recognized cause of infectious diarrhea inhospitalized patients. Enzyme immunoassay (EIA) testing has lower sensitivity relative toother methods, yet it has become commonplace due to cost. Sensitivity of ELISA testing isabout 60-90%, while newer molecular assays are about 95%. Some have suggested utilityin testing multiple samples by EIA to circumvent the lower sensitivities, especially in C.difficile associated diarrhea (CDAD) epidemics, but others have found limited data to supportthis. Repeated testing also delays diagnosis. The objective of this study was to determinethe cost-benefit of testing inpatients with a less sensitive EIA-based test for C. difficile toxinversus a more sensitive molecular assay. Methods: A retrospective chart review was performedof inpatients diagnosed with CDAD by EIA at our institution during an 11 month period.Those with previous diagnosis of CDAD within 30 days were excluded. Patients were dividedbetween those diagnosed with first EIA test versus multiple, and median hospital costsobtained. This analysis assumes that EIA testing more often relies on multiple testing, whilemolecular assays do not. Cost drivers were documented: need for consult, endoscopy, CTscan, abdominal ultrasound. A clinical assessment, per recent guidelines, of CDAD severitywas made. Additionally, risk factors for infection were documented: length of stay prior toinfection, age, antibiotic use within 14 days, previous CDADmore than 30 days prior, type ofantibiotic for CDAD, immunosuppression (chemotherapy, immunosuppressive medication,ESRD, DM), tube feeding, acid blocking medication, inflammatory bowel disease, recentbowel preparation or surgery. The incremental cost difference between the groups wasdetermined. Statistical differences in each category of clinical data were examined. A hypothet-ical annual savings was determined, assuming diagnosis on the first sample in all patients.Results: 156 patients met criteria. Of these, 101 were diagnosed after 1 EIA test, and 55were diagnosed after multiple. The median incremental cost increase for those requiringmultiple tests prior to diagnosis with CDAD was $48,356 per patient, or a total of about$2.66 million over this period. However, all cost drivers and nearly all clinical risk factorswere equally distributed. Only DM was found to be significantly greater in the grouprequiring multiple EIA tests. Conclusion: This cost-benefit analysis supports the hypothesisthat significant cost savings are achieved for inpatients diagnosed with CDAD with highersensitivity testing techniques rather than EIA. Adjusted annually at our institution, theseaccrued annual savings are 2.9 million dollars. Use of newer molecular assays, with farsuperior sensitivity, may be able to realize most of the potential savings.
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