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Inflammatory Profile Of Human Vocal Fold Fibroblasts In Response To Cigarette Smoke Extract
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS absent. Methods: Following IACUC approval, 8 mm full-thickness wounds were created on the dorsum of 8-12 week-old male C57/BL6 wild-type and ICAM-1 -/- mice. Pictures were taken daily to calculate wound-area. Tissues were harvested on post-injury days 1, 3, 7, and 14 (n¼6 per group) for histology. Samples from wound margins were evaluated for mRNA expression of 13 inflammatory cytokines on days 1 and 3 using RT-PCR. A separate group of wild-type and ICAM-1 -/- mice underwent a full-thickness paraspinal incision (n¼24), and tensiometric parameters were measured at 14 days post-injury. Finally, an unwounded group of wild-type mice (n¼4) and ICAM -/- mice (n¼4) underwent complete blood counts. Results: As compared with wild-type, ICAM-1 -/- wounds demonstrated inflamed, edematous margins with significantly delayed wound-closure through post-injury day 3 on histology (p<0.05) and day 8 as measured by wound-area (p<0.05) . There was significantly lower mRNA expression of KC and VEGF in the ICAM-1 -/- group as compared with wild-type on day 3 (p<0.05). There were no other differences in mRNA expression. ICAM -/- wounds had decreased elasticity as tensiometric studies showed significantly higher stiffness (18806926 kPa) compared to wild-type (4786117 kPa, p<0.01). ICAM-1 -/- showed significant lymphocytosis (7,130 vs. 4,740 cells/microliter, p<0.05), granulocytosis (2,130 vs. 630 cells/microliter, p<0.01), and overall leukocytosis (10,009 vs. 5,720 cells/microliter, p<0.05) compared with wild-type. Conclusions: ICAM-1 -/- mice exhibit a compensatory leukocytosis at baseline. The open wounds heal with an initial lag phase but catch-up to wild-type by day 8. Incisional wounds heal with significantly decreased elasticity at 14 days. Expression of many inflammatory cytokines is unchanged from wildtype. Interestingly, although this study displays delayed woundclosure, it demonstrates earlier normalization as compared with previous ICAM-1 knockout models, suggesting that sICAM-1 may also play a role in delaying wound-healing.
30.7. The Synergistic Effect of Pasireotide and a Raf-1 Activating Agent in Carcinoids. Y. R. Somnay, H. Chen, M. Kunnimalaiyaan; University of Wisconsin, Madison, WI Introduction: Somatostatin analogs are the mainstay treatment for controlling tumor proliferation and hormone secretion in carcinoid patients. The new somatostatin analog Pasireotide (SOM230) is thought to be a more effective drug given its broader receptor spectrum and elevated binding affinity relative to other somatostatin analogs. Recent data suggest that ERK1/2 phosphorylation may potentiate the anti-tumor effects of somatostatin analogs in carcinoids. Additionally, ERK1/2 phosphorylation by Raf-1 activating agents has been shown to suppress biomarker expression in carcinoids. Thus, drugs that activate the Raf-1/MEK/ERK1/2 pathway may be synergistic with somatostatin analogs such as SOM230. Here, we investigate the effects of SOM230 in combination with Teriflunomide (TFN), a Raf-1 activator, in a human carcinoid cell line. Methods: Human GI carcinoid cells (BON) were incubated in either TFN (0-125mM), SOM230 (0-10mM) or a combination, for 96 hours. Cell growth was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. Western analysis showed expression levels of achaete-scute complex-like1 (ASCL1) and Chromogranin A (CgA), well characterized markers for carcinoid malignancy. Growth inhibition mechanism was determined using Western analysis for phosphorylated and total ERK1/2, poly(ADP)-ribose polymerase (PARP), and other markers for apoptosis. Results: Combination treatment with SOM230 and TFN reduced cell growth beyond the additive effect of each drug alone. Combination index calculations strongly indicated synergy between both drugs. Treatment with TFN alone reduced ASCL1 and CgA expression in a dose dependent manner, by approximately 20% and 50% with 35mM and 50mM TFN respectively relative to control, while SOM230 alone had no affect. Notably, addition of SOM230 following these TFN doses further inhibited ASCL1 and CgA levels beyond the sum inhibitory effect of either drug alone. Combination of 0.5mM SOM230 and 50mM TFN reduced ASCL1 and
257
CgA levels by over 95% and 75% respectively, compared to no treatment. Additionally, combination treatment markedly increased levels of phosphorylated ERK1/2, cleaved PARP and cleaved caspase-3, and further reduced levels of total caspase-3, X-linked inhibitor of apoptosis (XIAP), survivin and Mcl-1, relative to treatment with either drug alone. Conclusions: This study offers compelling evidence that combination treatment with SOM230 and the Raf-1 activator TFN in BON carcinoid cells synergistically inhibits both cell growth and biomarker expression. Evidence of PARP and caspase-3 cleavage and inhibition of XIAP, survivin and Mcl-1 proves an apoptotic mechanism of synergy between these agents. Since treatment efficacy is achieved at lower doses than necessary with drug alone, combination therapy can accomplish symptomatic relief in carcinoid patients at tolerable toxicity levels. As each drug has already been evaluated independently in clinical trials, combinatorial drug trials are warranted.
30.8. Inflammatory Profile Of Human Vocal Fold Fibroblasts In Response To Cigarette Smoke Extract. A. R. Coughlin, C. M. Berchtold, S. Thibeault; University of WisconsinMadison, Madison, WI Introduction: Tobacco smoke is the dominant risk for laryngeal squamous neoplasia and chronic laryngitis. The hallmark of many such laryngeal diseases is the onset of mucosal inflammation, which can affect the critical functions of swallowing, breathing, and voice. While we currently have a limited understanding of how mucosal inflammation is manifested in response to cigarette smoke, we know that tissue specific fibroblasts are able to mediate chronic inflammation of other tissues through the secretion of soluble cytokines and paracrine regulation. Ultimately these processes can influence the outcomes of diseases such as cancer. In Reinke’s edema of the vocal fold, lamina propria fibroblasts are the primary cell type exposed to the inflammation induced edema associated with cigarette smoke exposure. Here we profile the cytokine response of human vocal fold fibroblasts to cigarette smoke exposure in vitro. Methods: Cigarette smoke extract (CSE) was prepared in accord with investigators studying the effects of cigarette smoke in vitro. One Marlboro cigarette with a removed filter was combusted into a 30 mL syringe at a rate of 3 extractions/minute over 5 minutes. The smoke was bubbled into 10 mL of cell culture media. This CSE was neutralized to pH 7.4, filtered,
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
adjusted to OD 1.0 (320nm) with a spectrophotometer and denoted 10% CSE. The control for CSE was air bubbled into the media. TNF-a (10 ng/mL) was used as a proinflammatory cytokine control. Immortalized human vocal fold fibroblasts (hVFF) were transiently treated for 3 hours with 0.5 and 1% CSE before being washed 2 times with 1x PBS and then returned to normal media for 24 hours. Trypan blue exclusion determined that cell death did not occur with CSE treatment. Cytokine levels of the media were assessed by the BioRad Bio-Plex 200 assay reader. Results: CSE (1.0%) had a significant increase in VEGF levels compared to control, CSE (0.5%) and TNFa (TABLE). TNF-a treatment showed a significant increase in IL-8 and cytokine TNF-a levels compared to control and CSE exposure. Although CSE (1.0%) exposure did show the trend of increasing IL-8 levels, this increase was not statistically significant. Additionally, IL-1B, IL-12 and Eotoxin levels did not change. Conclusions: Transient exposure of 1/10 of a combusted cigarette (CSE, 1.0%) showed the specific secretion of the angiogenesis regulatory cytokine VEGF relative to other treatments. Therefore, hVFFs could participate in the angiogenesis necessary for inflammation through the paracrine regulation of other tissues in the larynx. Lastly, since fibroblasts can mediate the inflammation associated with certain diseases, altering the immunomodulatory response of hVFFs could prevent diseases associated with mucosal inflammation of the larynx.
30.9. Dendritic Cells Are Necessary For Pancreas Viability In Acute Pancreatitis. A. H. Nguyen,1 A. Bedrosian,1 J. Henning,1 M. K. Connolly,1 A. Malhotra,2 N. Cieza-Rubio,1 J. Ibrahim,1 M. Marr,1 R. Barilla,2 M. Rapp,2 D. Ayo,1 P. Tan,1 H. Mushlin,1 I. C. Berkowitz,1 S. M. Cohen,1 G. Miller1,2; 1 Department of Surgery, New York University School of Medicine, New York, NY; 2Department of Cell Biology, New York University School of Medicine, New York, NY Introduction: Acute pancreatitis is characterized by an influx of inflammatory cell into the pancreas including dendritic cells (DC). However, the role of DC in pancreatitis is unknown. Since DC are central to initiating both robust inflammation and immune tolerance, we postulated that DC would have a primary role in regulating pancreatic insult. Methods: Cerulein and L-Arginine models of acute pancreatitis were employed in both C57BL/6 and CD11c-DTR mice in which DC depletion is accomplished by administration of diphtheria toxin. Tissue necrosis was determined by light microscopy using a computerized grid. Pancreatic infiltrates were assessed by flow cytometry. Cytokine levels were measured in a cytometric bead array. Survival was determined by the Kaplan-Meier method. Results: DC depleted mice challenged with cerulein developed necrosis of 96% pancreatic acini. Conversely, only 1% of pancreatic acini of mock depleted mice developed tissue necrosis after cerulein challenge (P<0.0001).
Furthermore, all mock depleted mice survived to 14 days, while 100% of DC depleted mice died within 5 days of cerulein challenge (P<0.0001). Similar effects of DC depletion were seen in an L-arginine model of acute pancreatitis. DC depletion was associated with elevated pancreatic expression of IL-6 and EGR-1 and serum TNF-a. DC depleted pancreata had a 10-fold increase in neutrophils and inflammatory monocytes after pancreatitis induction; however, tissue necrosis was independent of these cells. Similarly, IL-6, TNF-a, and NF-kB blockade did not mitigate the necrotic effects of DC depletion. Conclusions: These data demonstrate that DC are required for pancreas viability in acute pancreatitis.
30.10. A TLR4-MCP-1-macrophage IL-18 Cascade Plays A Major Role In Myocardial Injury And Cardiac Dysfunction After Permanent Ischemia. F. G. Al-amran, N. G. Yousif, X. M. Meng; Colorado University, Aurora, Co Introduction: Myocardial ischemia induces an inflammatory response involving in up-regulated chemokine expression and macrophage (Mf) accumulation. Toll-like receptor 4 (TLR4) has been linked to post-ischemic myocardial injury, and it mediates myocardial expression of MCP-1, a key chemokine for Mfaccumulation, after ischemia. However, the role of Mfin post-ischemic myocardial injury and heart failure is not well understood. We hypothesize that TLR4 mediates myocardial injury through up-regulation of chemokine expression and macrophage accumulation. The purposes of this study were to determine: 1) the effect of TLR4 mutation on different chemokines expression, differential leukocytes accumulation, and myocardial injury in a mouse model of permanent, 2) whether neutralizing MCP-1 reduces Mfaccumulation and myocardial injury, 3) whether monocyte/Mfdepletion reduces myocardial injury and 4)to determine the role of IL-18 expressed by accumulated macrophage, in the myocardial injury. Methods: C3H/HeJ (TLR4 mutant, with dysfunctional TLR4) and C3H/HeN (TLR4-competent) mice were subjected to left coronary ligation. Results: In TLR4-competent mice, among chemokines only MCP-1 myocardial levels increased markedly at day 1, 3 and 7, and massive Mfaccumulation in the ischemic myocardium occurred at day 3 and 7. TLR4 mutation significantly reduced these changes, resulting in smaller infarct size and improved heart function. Neutralizing MCP-1 also reduced Mfaccumulation, infarct size and heart failure in TLR4-competent mice. Further, prior depletion of monocytes/Mfusing liposomal clodronate resulted in infarct size reduction and heart function improvement. Finally, suppression of Mfdecreased myocardial IL-18 levels in ischemic myocardium, and IL-18 knockout provided significant myocardial protection against injury and dysfunction but its effect was less robust than TLR4 mutation, MCP-1 neutralization or monocytes/Mfdepletion. Conclusions: A TLR4-MCP-1-macrophage-IL-18 cascade occupies a major in myocardial injury and cardiac dysfunction after permanent ischemia and could be a therapeutic target for protection against post-ischemic myocardial injury and heart failure.
SESSION: WEDNESDAY, FEB 2, 2011 1:30 - 4:00 PM AAS PLENARY SESSION 31.1. Anti-CA19-9 Diabody As A PET Imaging Probe For Pancreas Cancer. M. D. Girgis,1 V. Kenanova,2 T. Olafsen,2 K. E. McCabe,2 F. Bergara,2 A. M. Wu,2 J. S. Tomlinson1; 1 Department of Surgery, University of California, Los Angeles, Los Angeles, CA; 2Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, CA
30.7. The Synergistic Effect of Pasireotide and a Raf-1 Activating Agent in Carcinoids. Y. R. Somnay, H. Chen, M. Kunnimalaiyaan; University of Wisconsin, Madison, WI Introduction: Somatostatin analogs are the mainstay treatment for controlling tumor proliferation and hormone secretion in carcinoid patients. The new somatostatin analog Pasireotide (SOM230) is thought to be a more effective drug given its broader receptor spectrum and elevated binding affinity relative to other somatostatin analogs. Recent data suggest that ERK1/2 phosphorylation may potentiate the anti-tumor effects of somatostatin analogs in carcinoids. Additionally, ERK1/2 phosphorylation by Raf-1 activating agents has been shown to suppress biomarker expression in carcinoids. Thus, drugs that activate the Raf-1/MEK/ERK1/2 pathway may be synergistic with somatostatin analogs such as SOM230. Here, we investigate the effects of SOM230 in combination with Teriflunomide (TFN), a Raf-1 activator, in a human carcinoid cell line. Methods: Human GI carcinoid cells (BON) were incubated in either TFN (0-125mM), SOM230 (0-10mM) or a combination, for 96 hours. Cell growth was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) rapid colorimetric assay. Western analysis showed expression levels of achaete-scute complex-like1 (ASCL1) and Chromogranin A (CgA), well characterized markers for carcinoid malignancy. Growth inhibition mechanism was determined using Western analysis for phosphorylated and total ERK1/2, poly(ADP)-ribose polymerase (PARP), and other markers for apoptosis. Results: Combination treatment with SOM230 and TFN reduced cell growth beyond the additive effect of each drug alone. Combination index calculations strongly indicated synergy between both drugs. Treatment with TFN alone reduced ASCL1 and CgA expression in a dose dependent manner, by approximately 20% and 50% with 35mM and 50mM TFN respectively relative to control, while SOM230 alone had no affect. Notably, addition of SOM230 following these TFN doses further inhibited ASCL1 and CgA levels beyond the sum inhibitory effect of either drug alone. Combination of 0.5mM SOM230 and 50mM TFN reduced ASCL1 and
257
CgA levels by over 95% and 75% respectively, compared to no treatment. Additionally, combination treatment markedly increased levels of phosphorylated ERK1/2, cleaved PARP and cleaved caspase-3, and further reduced levels of total caspase-3, X-linked inhibitor of apoptosis (XIAP), survivin and Mcl-1, relative to treatment with either drug alone. Conclusions: This study offers compelling evidence that combination treatment with SOM230 and the Raf-1 activator TFN in BON carcinoid cells synergistically inhibits both cell growth and biomarker expression. Evidence of PARP and caspase-3 cleavage and inhibition of XIAP, survivin and Mcl-1 proves an apoptotic mechanism of synergy between these agents. Since treatment efficacy is achieved at lower doses than necessary with drug alone, combination therapy can accomplish symptomatic relief in carcinoid patients at tolerable toxicity levels. As each drug has already been evaluated independently in clinical trials, combinatorial drug trials are warranted.
30.8. Inflammatory Profile Of Human Vocal Fold Fibroblasts In Response To Cigarette Smoke Extract. A. R. Coughlin, C. M. Berchtold, S. Thibeault; University of WisconsinMadison, Madison, WI Introduction: Tobacco smoke is the dominant risk for laryngeal squamous neoplasia and chronic laryngitis. The hallmark of many such laryngeal diseases is the onset of mucosal inflammation, which can affect the critical functions of swallowing, breathing, and voice. While we currently have a limited understanding of how mucosal inflammation is manifested in response to cigarette smoke, we know that tissue specific fibroblasts are able to mediate chronic inflammation of other tissues through the secretion of soluble cytokines and paracrine regulation. Ultimately these processes can influence the outcomes of diseases such as cancer. In Reinke’s edema of the vocal fold, lamina propria fibroblasts are the primary cell type exposed to the inflammation induced edema associated with cigarette smoke exposure. Here we profile the cytokine response of human vocal fold fibroblasts to cigarette smoke exposure in vitro. Methods: Cigarette smoke extract (CSE) was prepared in accord with investigators studying the effects of cigarette smoke in vitro. One Marlboro cigarette with a removed filter was combusted into a 30 mL syringe at a rate of 3 extractions/minute over 5 minutes. The smoke was bubbled into 10 mL of cell culture media. This CSE was neutralized to pH 7.4, filtered,
258
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
adjusted to OD 1.0 (320nm) with a spectrophotometer and denoted 10% CSE. The control for CSE was air bubbled into the media. TNF-a (10 ng/mL) was used as a proinflammatory cytokine control. Immortalized human vocal fold fibroblasts (hVFF) were transiently treated for 3 hours with 0.5 and 1% CSE before being washed 2 times with 1x PBS and then returned to normal media for 24 hours. Trypan blue exclusion determined that cell death did not occur with CSE treatment. Cytokine levels of the media were assessed by the BioRad Bio-Plex 200 assay reader. Results: CSE (1.0%) had a significant increase in VEGF levels compared to control, CSE (0.5%) and TNFa (TABLE). TNF-a treatment showed a significant increase in IL-8 and cytokine TNF-a levels compared to control and CSE exposure. Although CSE (1.0%) exposure did show the trend of increasing IL-8 levels, this increase was not statistically significant. Additionally, IL-1B, IL-12 and Eotoxin levels did not change. Conclusions: Transient exposure of 1/10 of a combusted cigarette (CSE, 1.0%) showed the specific secretion of the angiogenesis regulatory cytokine VEGF relative to other treatments. Therefore, hVFFs could participate in the angiogenesis necessary for inflammation through the paracrine regulation of other tissues in the larynx. Lastly, since fibroblasts can mediate the inflammation associated with certain diseases, altering the immunomodulatory response of hVFFs could prevent diseases associated with mucosal inflammation of the larynx.
30.9. Dendritic Cells Are Necessary For Pancreas Viability In Acute Pancreatitis. A. H. Nguyen,1 A. Bedrosian,1 J. Henning,1 M. K. Connolly,1 A. Malhotra,2 N. Cieza-Rubio,1 J. Ibrahim,1 M. Marr,1 R. Barilla,2 M. Rapp,2 D. Ayo,1 P. Tan,1 H. Mushlin,1 I. C. Berkowitz,1 S. M. Cohen,1 G. Miller1,2; 1 Department of Surgery, New York University School of Medicine, New York, NY; 2Department of Cell Biology, New York University School of Medicine, New York, NY Introduction: Acute pancreatitis is characterized by an influx of inflammatory cell into the pancreas including dendritic cells (DC). However, the role of DC in pancreatitis is unknown. Since DC are central to initiating both robust inflammation and immune tolerance, we postulated that DC would have a primary role in regulating pancreatic insult. Methods: Cerulein and L-Arginine models of acute pancreatitis were employed in both C57BL/6 and CD11c-DTR mice in which DC depletion is accomplished by administration of diphtheria toxin. Tissue necrosis was determined by light microscopy using a computerized grid. Pancreatic infiltrates were assessed by flow cytometry. Cytokine levels were measured in a cytometric bead array. Survival was determined by the Kaplan-Meier method. Results: DC depleted mice challenged with cerulein developed necrosis of 96% pancreatic acini. Conversely, only 1% of pancreatic acini of mock depleted mice developed tissue necrosis after cerulein challenge (P<0.0001).
Furthermore, all mock depleted mice survived to 14 days, while 100% of DC depleted mice died within 5 days of cerulein challenge (P<0.0001). Similar effects of DC depletion were seen in an L-arginine model of acute pancreatitis. DC depletion was associated with elevated pancreatic expression of IL-6 and EGR-1 and serum TNF-a. DC depleted pancreata had a 10-fold increase in neutrophils and inflammatory monocytes after pancreatitis induction; however, tissue necrosis was independent of these cells. Similarly, IL-6, TNF-a, and NF-kB blockade did not mitigate the necrotic effects of DC depletion. Conclusions: These data demonstrate that DC are required for pancreas viability in acute pancreatitis.
30.10. A TLR4-MCP-1-macrophage IL-18 Cascade Plays A Major Role In Myocardial Injury And Cardiac Dysfunction After Permanent Ischemia. F. G. Al-amran, N. G. Yousif, X. M. Meng; Colorado University, Aurora, Co Introduction: Myocardial ischemia induces an inflammatory response involving in up-regulated chemokine expression and macrophage (Mf) accumulation. Toll-like receptor 4 (TLR4) has been linked to post-ischemic myocardial injury, and it mediates myocardial expression of MCP-1, a key chemokine for Mfaccumulation, after ischemia. However, the role of Mfin post-ischemic myocardial injury and heart failure is not well understood. We hypothesize that TLR4 mediates myocardial injury through up-regulation of chemokine expression and macrophage accumulation. The purposes of this study were to determine: 1) the effect of TLR4 mutation on different chemokines expression, differential leukocytes accumulation, and myocardial injury in a mouse model of permanent, 2) whether neutralizing MCP-1 reduces Mfaccumulation and myocardial injury, 3) whether monocyte/Mfdepletion reduces myocardial injury and 4)to determine the role of IL-18 expressed by accumulated macrophage, in the myocardial injury. Methods: C3H/HeJ (TLR4 mutant, with dysfunctional TLR4) and C3H/HeN (TLR4-competent) mice were subjected to left coronary ligation. Results: In TLR4-competent mice, among chemokines only MCP-1 myocardial levels increased markedly at day 1, 3 and 7, and massive Mfaccumulation in the ischemic myocardium occurred at day 3 and 7. TLR4 mutation significantly reduced these changes, resulting in smaller infarct size and improved heart function. Neutralizing MCP-1 also reduced Mfaccumulation, infarct size and heart failure in TLR4-competent mice. Further, prior depletion of monocytes/Mfusing liposomal clodronate resulted in infarct size reduction and heart function improvement. Finally, suppression of Mfdecreased myocardial IL-18 levels in ischemic myocardium, and IL-18 knockout provided significant myocardial protection against injury and dysfunction but its effect was less robust than TLR4 mutation, MCP-1 neutralization or monocytes/Mfdepletion. Conclusions: A TLR4-MCP-1-macrophage-IL-18 cascade occupies a major in myocardial injury and cardiac dysfunction after permanent ischemia and could be a therapeutic target for protection against post-ischemic myocardial injury and heart failure.
SESSION: WEDNESDAY, FEB 2, 2011 1:30 - 4:00 PM AAS PLENARY SESSION 31.1. Anti-CA19-9 Diabody As A PET Imaging Probe For Pancreas Cancer. M. D. Girgis,1 V. Kenanova,2 T. Olafsen,2 K. E. McCabe,2 F. Bergara,2 A. M. Wu,2 J. S. Tomlinson1; 1 Department of Surgery, University of California, Los Angeles, Los Angeles, CA; 2Crump Institute for Molecular Imaging, University of California, Los Angeles, Los Angeles, CA