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Site-1-Protease Mediated Unfolded Protein Response Protects Against Pancreatic Injury in Acute Pancreatitis
AGA Abstracts
and α-SMA, as measured on IHC, decreased significantly after naltrexone treatment. Conclusions: Our data clearly shows that naltrexone treatment protects against progression of chronic pancreatitis as well as reduced severity of established CP. Given that naltrexone is already in clinical use it could potentially emerge as novel disease modifying therapeutic strategy against chronic pancreatitis
Tu1353 PATHOGENESIS OF CHRONIC PANCREATITIS IS INDEPENDENT OF TRYPSIN ACTIVATION IN L-ARGININE INDUCED MOUSE MODEL OF DISEASE John George, Ajay Dixit, Hassam Cheema, Bhuwan Giri, Vikas Dudeja, Rajinder Dawra, Ashok Saluja BACKGROUND & AIMS: Pathogenic mechanisms leading to inflammation and fibrosis in chronic pancreatitis are unclear. We have previously shown that while acinar cell injury during acute pancreatitis(AP) is mediated by trypsin, inflammation during AP is independent of trypsin activation. Conventionally, trypsin is believed to be central event in chronic pancreatitis as well (CP). Since inflammation is the key component of CP and since inflammation during AP is independent of trypsin, in the current study we have evaluated the role of trypsin in pathogenesis of CP in L-arginine induced mouse model of CP. METHODS: L-Arginine CP was induced in in wild-type, Trypsinogen 7-KO [T (-/-)], and Cathepsin B knock out [CB (-/-)] mice. In this model L-Arginine AP is induced weekly, for 4 weeks and the animals were sacrificed at 7 days after the last cycle of AP. Markers of CP including pancreatic atrophy (as measured by pancreas to mouse weight ratio), histopathology, collagen content (Sirius red staining), inflammation were measured and compared between groups. RESULTS: Pancreas to mouse weight ratio was significantly reduced in L-arginine CP group when compared to control mice (3.2±0.3 vs 9.2±0.4) suggesting pancreatic atrophy. Pancreas to mouse weight ratio in T (-/-) (3.4±0.2) and CB (-/-) (3.1±0.2) knockout mice was not significantly different from that of WT mice with L-arginine CP, suggesting that pancreatic atrophy did not change with lack of trypsin or cathepsin B, which is required for trypsin activation. The acinar cell loss, chronic inflammatory infiltrate and fibro fatty replacement induced by induction of L-arginine CP was similar in WT, T (-/-) and CB (-/-) mice. Similarly, collagen deposition as measured by Sirius red staining was not different between WT, T (-/-) and CB (-/-) mice. Chronic inflammatory infiltrate, measured by leukocytic IHC for CD45, showed similar degree of inflammation in WT, trypsin and cathepsin B knockout mice. We also observed significant steatorrhea in all the groups of L-Arginine pancreatitis suggestive of progressive exocrine insufficiency. CONCLUSIONS: In L-arginine model of CP, absence of trypsin or cathepsin B did not modulate or attenuate severity of chronic pancreatitis. This may suggest that premature trypsin activation, which is believed to be the mechanism of injury in hereditary pancreatitis, may lead to chronic pancreatitis but is not a requisite for development of CP.
Tu1355 ACTIVIN IN ACUTE PANCREATITIS: POTENTIAL RISK-STRATIFYING MARKER AND NOVEL THERAPEUTIC TARGET Jonas J. Staudacher, Cemal Yazici, Timothy Carroll, Nancy Krett, Jessica I. Bauer, Yinglin Xia, Jingbo Pang, Annette Wilson, Georgios I. Papachristou, David C. Whitcomb, Giamila Fantuzzi, Barbara Jung Background and Aims: Acute Pancreatitis is a substantial health care challenge with increasing incidence. Patients who develop severe disease have considerable mortality. Currently, no reliable predictive marker to identify patients at risk for severe disease exists. Treatment is limited to rehydration and supporting care suggesting an urgent need to develop novel approaches to improve standard care. Activin is a critical modulator of inflammatory responses, but has not been assessed in pancreatitis. Methods: Two established murine models of acute pancreatitis induced by either cerulein or IL-12+IL-18 were evaluated for serum activin levels measured over time and correlated with disease severity. In addition, serum activin levels were measured from a retrospective clinical cohort of pancreatitis patients and correlated with overall survival and mortality as well as rate of intensive care unit admission and length of hospital stay. Results: Here, we demonstrate that serum activin is elevated and strongly correlates with disease severity in two preclinical models and a human cohort of acute pancreatitis. Furthermore, in mice, inhibition of activin conveys survival benefits at early time points in pancreatitis, and high activin levels in patients at admission are predictive of worse outcomes, indicated by longer overall hospital and intensive care unit stays. Conclusions: Taken together, activin is a novel candidate as a clinical marker and therapeutic target in acute pancreatitis.
Tu1354 SIMULTANEOUS ACTIVATION OF K-RAS AND NRF2 INDUCE PANCREATIC ATROPHY Shin Hamada, Atsushi Masamune, Keiko Taguchi, Masayuki Yamamoto, Tooru Shimosegawa Background: The Keap1-Nrf2 system plays pivotal roles that induce wide variety of cytoprotective genes responsible for cell proliferation and metabolic reprogramming. Aberrant Nrf2 activation is frequently observed in cancer cells, yielding growth and survival advantages in the progression. To clarify the role of Nrf2 in pancreatic carcinogenesis using mutant K-ras and p53 mouse model, we examined if Nrf2 activated by Keap1 deletion facilitates the cancer. Materials and methods: LSL-K-rasG12D, LSL-p53R172H, and Pdx-1-Cre mice were crossed with Keap1fl/fl and/or Nrf2-/- mice to generate mice with genotypes summarized in figure 1. Mice were treated according to the Guidelines for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science and Technology of Japan. These mice were sacrificed at day 90, unless they reached a humane endpoint earlier. Body weight of mice was checked weekly after birth. Serum amylase and glucose were measured at the time of sacrifice. Expression of amylase, insulin, α-smooth muscle actin (α-SMA) and Nqo1 were assessed by immunohistochemistry. Results: Regardless of the presence or absence of p53 loss, mice with K-ras mutant and Keap1 deletion resulted in pancreatic atrophy, which shortened survival time. These mice revealed body weight loss and hypoglycemia, indicating malnutrition. In pancreas with K-ras mutant and Keap1 deletion, Nrf2 activation was evaluated by the elevated expression of typical Nrf2-target gene Nqo1. Amylase positive acinar cells and insulin positive islet cells were reduced in the pancreas of mice with K-ras mutant and Keap1 deletion. Degenerative acinar cells and islet cells were surrounded by α-SMA positive stromal cells, suggesting fibrosis. This phenotype was dependent on Nrf2, because Nrf2 deletion rescued the phenotype in K-ras mutant and Keap1 deletion. Discussion: Pancreas-specific activation of K-ras and Nrf2 caused malnutrition due to loss of pancreatic parenchyma, rather than acceleration of carcinogenesis (figure 2). Nrf2 activation in the context of K-ras mutation might have alternative roles.
AGA Abstracts
Tu1356 SITE-1-PROTEASE MEDIATED UNFOLDED PROTEIN RESPONSE PROTECTS AGAINST PANCREATIC INJURY IN ACUTE PANCREATITIS Kaylene Barrera, Kei Okochi, Albert Stanek, Cathy Mueller, Peiqi Ou, Antonio Alfonso, Chongmin Huan Introduction: Acute pancreatitis (AP) mainly involves acinar cell injury that is initiated when cellular self-protection is overwhelmed by pathogenic dysregulation. Although premature intracellular activation of trypsinogen was widely thought to induce AP, recent studies show that endoplasmic reticulum (ER) stress triggered by multiple AP predisposing factors leads to acinar injury if not salvaged by the unfolded protein response (UPR). Site-1-protease (S1P) is a Golgi membrane-bound UPR mediator that enables activating transcription factor 6 (ATF6) to activate transcription of UPR-target genes via cleavage activation of ATF6. But the self-protective role of S1P-mediated UPR in AP is unknown. Here we study cerulein AP and ethanol-induced acinar abnormalities in S1P deficient mice. Methods: Paired S1Pflox/+ and S1Pflox/flox littermates that express normal level and 70% less pancreatic S1P respectively were studied. Cerulein AP was induced via 7 hourly cerulein (50µg/kg) intraperitoneal injections. Ethanol was administrated by following a binge-like alcohol intake protocol. Pancreatic injury was analyzed by histological and biochemistry studies. Isolated S1Pflox/+ and S1Pflox/flox acinar cells were cultured with ethanol or cerulein for measurement of ER stress-induced S1P mRNA and nuclear ATF6 levels by RT-PCR and Western blot respectively. Results: Cerulein and ethanol treatment increased S1P expression and nuclear ATF6 in acinar cells. Compared to S1Pflox/+ mice, S1Pflox/flox mice had significantly increased serum amylase and lipase, pancreatic acinar injury and inflammatory infiltration in cerulein AP (Figure 1). Consistently, ethanol intake caused higher serum amylase levels and more abnormal pancreatic acinar cells in S1Pflox/flox mice compared to S1Pflox/+ mice (Figure 2). Conclusion: Induced S1P activity in AP protects against acinar cell injury.
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Cerulein induced acute pancreatitis. (L) S1Pflox/+ (R) S1P flox/flox Tu1359 EFFECT OF OCTREOTIDE ON PANCREATIC FIBROSIS OF HIGH-FAT DIET INDUCED OBESITY IN RATS Ting Ye, Rui Liu Objective High-fat diets can induce inflammatory response of pancreas that ultimately lead to pancreatic fibrosis, which is related to activation of pancreatic stellate cells (PSCs). Previous studies have suggested that somatostatin (SST) and its analogs were effective in treating hepatic fibrosis through inhibiting hepatic stellate cells specifically. The aim of this study was to investigate the effects of octreotide on experimental pancreatic fibrosis in obesity rats by high-fat diet (HFD) induced. Methods Sprague-Dawley (SD) male rats were randomly assigned to control, HFD, or octreotide-treated groups. Glucose and insulin tolerance test were determined then calculate the area under the curve (AUC) of the tests. Changes in pancreatic morphology; fasting plasma glucose (FPG) and fasting insulin (FINS); serum triglyceride (TG), total cholesterol (TC), free fatty acid (FFA); pancreatic TG were measured, and Lee's index and homeostatic model assessment (HOMA) index were also calculated. The expression levels of alpha-smooth muscle actin(α-SMA) , desmin, connective tissue growth factor(CTGF), transforming growth factor beta1(TGF-β1),Smad3, Smad7 in pancreas were quantified. Rat pancreatic stellate cells were divided into three groups: (1) control group; (2) palmitate (PA) group; (3)octreotide-treated group, and the expression of α-SMA, CTGF, TGF-β1,Smad3,Smad7 were quantified. Results Compared with the control group, body weight, Lee's index, AUC of ipGTT and ipITT, FPG, FINS, TG, TC and FFA levels, and HOMA index in HFD group were significantly increased. The values of some of these parameters were significantly reduced after octreotide treatment, while Lee's index, TC, TG levels, and AUC were tended to decrease but have no statistical significance. The expression levels of α-SMA, CTGF, TGF-beta1, Smad3 protein and mRNA were significantly higher and Smad7 protein and mRNA level were significantly lower in HFD group than those in the control group. After octreotide treatment, α-SMA, CTGF, TGF-β1, Smad3 protein and mRNA levels were decreased and Smad7 increased. In vitro study, we found that the expression levels of CTGF, TGF-β1, Smad3 protein and mRNA were increased in PA group, and octreotide-treatment down-regulated the expression levels of these molecules. The Smad7 and α-SMA protein and mRNA levels did not show significant changes in PA group or in octreotide-adminstrated group. Conclusion Octreotide can ameliorate pancreatic fibrosis and improve the pancreatic beta-cell function in HFD induced rats, possibly via inhibiting PSC activation and decreasing the pancreatic extracellular matrix (ECM) through TGF-β1/Smad signaling pathway.
Alcohol binge drinking model. (L)S1Pflox/+ (R) S1Pflox/flox
Tu1357 NOVEL SMALL MOLECULE PKD INHIBITORS SUPPRESS NF-ΚB ACTIVATION, INFLAMMATION, NECROSIS AND SEVERITY IN PANCREATITIS Jingzhen Yuan, Meng Geng, Tanya Tan, Grace Tan, Stephen J. Pandol Background and Aims: Acute pancreatitis is characterized by inflammation and necrosis of the parenchymal cells in the pancreas. The pro-inflammatory transcription factor NF-κB plays a critical role in regulation of inflammatory and cell death responses. We previously reported that protein kinase D (PKD) regulates NF-κB activation induced by CCK8 or carbachol in pancreatic acinar cell line AR42J cells. With pharmacological approach, we further showed that NF-κB activation was markedly suppressed by two novel small molecule PKD inhibitors in experimental models of acute pancreatitis, demonstrating that PKD is an important mediator of NF-κB activation in pancreatitis. The aim of the current studies was to continually explore whether suppressing of NF-κB activation by the PKD inhibitors was associated with attenuation of inflammatory responses and severity of the disease in experimental model of pancreatitis. Methods: Sprague-Dawley rats were pre-treated with intraperitoneal (IP) injection of PKD inhibitor CID755673 (CID, 15mg/kg) or CRT0066101 (CRT, 10mg/kg) for 60 min prior to initiation of pancreatic stress with up to 4 hourly IP injections of cerulein (20mg/kg) or saline. Then the animals were sacrificed in 30 min after first and fourth hourly IP injection of cerulein. NF-κB-DNA binding activity in pancreatic tissue nuclear extracts was measured with electrophoretic mobility shift assay. Western blotting was performed to analyze PKD phosphorylation, NF-κB nucleus translocation, and pancreatic protein level of IL-6 with specific antibodies. Pancreatitis parameters were evaluated on H&E stained pancreatic tissue sections. Results: We found that either CID or CRT blocked cerulein-induced PKD autophosphorylation at Ser916 without affecting PKCdependent Ser744/748 phosphorylation in pancreatitis, demonstrating specificity of the PKD inhibitor. PKD inhibition resulted in markedly suppressed NF-κB-DNA binding activity in nuclear extracts and NF-κB nucleus translocation. Importantly, NF-κB inhibition by the PKD inhibitor CID was associated with significantly decreased inflammatory responses and necrosis indicated by reduced pancreatic IL-6 protein, less inflammatory cell infiltration and myeloperoxidase positively stained cells in pancreatic tissue sections, and improved pancreatic necrosis, edema and vacuolization. CONCLUSION: PKD is an important mediator of NF-κB activation in pancreatitis. NF-κB inhibition by specific PKD inhibitor was associated with decreased inflammatory responses and attenuated necrosis and pancreatitis severity in a model of acute pancreatitis. The results suggest that the novel small molecule PKD inhibitors have high potential as therapeutic agents to prevent recurrent pancreatitis through suppressing NF-κB activation.
Tu1360 EFFECTS OF UNSATURATED FREE FATTY ACIDS (UFFA) ON HUMAN ENDOTHELIAL CELLS: IS THERE A THRESHOLD UFFA LEVEL CAUSING CELL TOXICITY AND DEATH IN ACUTE PANCREATITIS? Anna C. Evans, Annette Wilson, Georgios I. Papachristou, David C. Whitcomb BACKGROUND: Hypertriglyceridemic (HTG) acute pancreatitis (AP) is often severe, with up to 30% mortality. While HTG itself is rather benign, release of pancreatic lipase(s) into the circulation during an episode of AP facilitates massive hydrolysis of TG to free fatty acids (FFA). In addition to the pathogenic mechanism of typical AP, release of linoleic acid (LA) and other uFFA causes direct toxicity in animal models. In humans, endothelial injury or stress results in a vascular leak syndrome (VLS), linking systemic inflammation with a multi-organ dysfunction syndrome. It is not known if uFFA affect human microvascular endothelial cells, and if any pathologic effect is concentration-dependent. Since two pathogenic processes are possible in HTG-AP (cytokine storm and lipotoxicity), it is important distinguish the two processes. Knowing the effect of uFFA on human endothelial cells is critical to solving this problem. HYPOTHESIS: We hypothesize that Linoleic Acid(LA) causes increased cell death of in human intestinal microvascular endothelial cells (HiMECs), thereby causing the disruption of endothelial barrier integrity, eventually resulting in vascular leak syndrome independent of cytokines. METHODS: Human intestinal microvascular epithelial cells were isolated from surgical margin specimens using CD31 marker beads. They were passaged in vitro and passages 5-15 were used for experimentation. Linoleic Acid (18:2) was dissolved in DMSO to create solutions of increasing concentration and applied directly to cells for 24h. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was tested using colorimetric assay. LDH (Lactate Dehydrogenase) was tested also using colorimetric assay. One-way ANOVA and post-hoc Tukey tests were used to determine significant differences. RESULTS: Endothelial cells treated with increasingly concentrated solutions of Linoleic Acid (18:2) have decreasing viability (Fig 1). Absorbance was measured as Optical Density (OD) and was read by a BioRad Microplate Reader with 570nM filter. Bars show average reading of optical density (n=18) with standard deviation. Significant differences (p<0.05) are seen between controls and concentrations of 1.25mM, 2.5mM, and 5.0mM Linoleic Acid. Increased levels of LDH were also found with increased concentrations of Linoleic Acid. Significant differences were found between cells treated w 2.5mM Linoleic Acid, 5.0mM Linoleic Acid, and controls. CONCLUSIONS: uFFA such as LA causes direct cell toxicity in human microvascular endothelial cells at concentrations at, or above 1.25 mM. Clinical studies in humans are needed to correlate uFFA levels and evidence of VLA and lipotoxicity, followed by trials to rapidly clear FFA when the concentration exceed the threshold for lipotoxicity.
Tu1358 LIPOTOXICITY MAY RESULT IN INFLAMMATORY CELL DEATH AND ASSOCIATED IMMUNE PARALYSIS DURING SEVERE ACUTE PANCREATITIS (SAP) Bara El Kurdi, Jordan R. Yaron, Shehryar Masood, Pawan Noel, Krutika Patel, Biswajit Khatua, Cristiane Oliveira, Vijay P. Singh Background: Patients with SAP can develop the Compensatory Anti-inflammatory Response syndrome (CARS), associated with opportunistic infections. Additionally, a White blood cell (WBC) count < 4000/mm3 is part of the criteria for Systemic Inflammatory Response Syndrome (SIRS) which can develop in SAP. This study was designed to understand these phenomena. Methods: The Mayo clinic autopsy database was searched for patients with who died with necrotizing pancreatitis (NP; n=8) or unrelated causes (controls). Age, gender, BMI were recorded and spleens of these were stained for apoptotic cells (TUNEL) and quantified as % positive nuclei. Additionally, SAP was induced in Wistar rats (250gm) by administration of caerulein (CER; 50mcg/kg BID) and glyceryl trioleate (GTO) alone or along with the lipase inhibitor orlistat (GTOO) as described previously [PMID 25500204]. WBC counts, fatty acids were quantified in the blood of these rats and TUNEL staining was done on their spleens and quantified as number of positive cells per white pulp pixel area. A p of <0.05 was regarded as significant. Results: The mean BMI of patients with NP was 45.6 kg/m2 vs. 26.1 kg/m2 in controls. NP was associated with an increase in TUNEL positive cells in the spleen (1% vs. 10%, p=0.0117) including the white pulp (1% vs. 10%, p= 0.0155). The CER+GTO group had 100% mortality (vs. 0% in all other groups). This was
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AGA Abstracts
AGA Abstracts
associated with a 6.4 fold increase in TUNEL positive cells in the white pulp of the spleens of rats in the CER+GTO group vs. controls (p<0.0001) and a decrease in total WBC count from (12.2±1.2×103/mm3 in control to 3.5±0.5×103/mm3, p<0.02), along with an increase in circulating free fatty acids (539±116µM vs 252±91µM in controls p<0.003). These changes were prevented in the CER+GTOO group, which was not significantly different from controls. Conclusion: CARS can result from increased inflammatory cell death during severe illnesses such as necrotizing pancreatitis. This may be seen as a reduction in circulating WBC counts and an increase in apoptotic inflammatory cells- as we note in the spleens during SAP. Targeting lipotoxicity form the fatty acids released from visceral fat lipolysis may reduce CARS during SAP and the immune paralysis associated with it.
and α-SMA, as measured on IHC, decreased significantly after naltrexone treatment. Conclusions: Our data clearly shows that naltrexone treatment protects against progression of chronic pancreatitis as well as reduced severity of established CP. Given that naltrexone is already in clinical use it could potentially emerge as novel disease modifying therapeutic strategy against chronic pancreatitis
Tu1353 PATHOGENESIS OF CHRONIC PANCREATITIS IS INDEPENDENT OF TRYPSIN ACTIVATION IN L-ARGININE INDUCED MOUSE MODEL OF DISEASE John George, Ajay Dixit, Hassam Cheema, Bhuwan Giri, Vikas Dudeja, Rajinder Dawra, Ashok Saluja BACKGROUND & AIMS: Pathogenic mechanisms leading to inflammation and fibrosis in chronic pancreatitis are unclear. We have previously shown that while acinar cell injury during acute pancreatitis(AP) is mediated by trypsin, inflammation during AP is independent of trypsin activation. Conventionally, trypsin is believed to be central event in chronic pancreatitis as well (CP). Since inflammation is the key component of CP and since inflammation during AP is independent of trypsin, in the current study we have evaluated the role of trypsin in pathogenesis of CP in L-arginine induced mouse model of CP. METHODS: L-Arginine CP was induced in in wild-type, Trypsinogen 7-KO [T (-/-)], and Cathepsin B knock out [CB (-/-)] mice. In this model L-Arginine AP is induced weekly, for 4 weeks and the animals were sacrificed at 7 days after the last cycle of AP. Markers of CP including pancreatic atrophy (as measured by pancreas to mouse weight ratio), histopathology, collagen content (Sirius red staining), inflammation were measured and compared between groups. RESULTS: Pancreas to mouse weight ratio was significantly reduced in L-arginine CP group when compared to control mice (3.2±0.3 vs 9.2±0.4) suggesting pancreatic atrophy. Pancreas to mouse weight ratio in T (-/-) (3.4±0.2) and CB (-/-) (3.1±0.2) knockout mice was not significantly different from that of WT mice with L-arginine CP, suggesting that pancreatic atrophy did not change with lack of trypsin or cathepsin B, which is required for trypsin activation. The acinar cell loss, chronic inflammatory infiltrate and fibro fatty replacement induced by induction of L-arginine CP was similar in WT, T (-/-) and CB (-/-) mice. Similarly, collagen deposition as measured by Sirius red staining was not different between WT, T (-/-) and CB (-/-) mice. Chronic inflammatory infiltrate, measured by leukocytic IHC for CD45, showed similar degree of inflammation in WT, trypsin and cathepsin B knockout mice. We also observed significant steatorrhea in all the groups of L-Arginine pancreatitis suggestive of progressive exocrine insufficiency. CONCLUSIONS: In L-arginine model of CP, absence of trypsin or cathepsin B did not modulate or attenuate severity of chronic pancreatitis. This may suggest that premature trypsin activation, which is believed to be the mechanism of injury in hereditary pancreatitis, may lead to chronic pancreatitis but is not a requisite for development of CP.
Tu1355 ACTIVIN IN ACUTE PANCREATITIS: POTENTIAL RISK-STRATIFYING MARKER AND NOVEL THERAPEUTIC TARGET Jonas J. Staudacher, Cemal Yazici, Timothy Carroll, Nancy Krett, Jessica I. Bauer, Yinglin Xia, Jingbo Pang, Annette Wilson, Georgios I. Papachristou, David C. Whitcomb, Giamila Fantuzzi, Barbara Jung Background and Aims: Acute Pancreatitis is a substantial health care challenge with increasing incidence. Patients who develop severe disease have considerable mortality. Currently, no reliable predictive marker to identify patients at risk for severe disease exists. Treatment is limited to rehydration and supporting care suggesting an urgent need to develop novel approaches to improve standard care. Activin is a critical modulator of inflammatory responses, but has not been assessed in pancreatitis. Methods: Two established murine models of acute pancreatitis induced by either cerulein or IL-12+IL-18 were evaluated for serum activin levels measured over time and correlated with disease severity. In addition, serum activin levels were measured from a retrospective clinical cohort of pancreatitis patients and correlated with overall survival and mortality as well as rate of intensive care unit admission and length of hospital stay. Results: Here, we demonstrate that serum activin is elevated and strongly correlates with disease severity in two preclinical models and a human cohort of acute pancreatitis. Furthermore, in mice, inhibition of activin conveys survival benefits at early time points in pancreatitis, and high activin levels in patients at admission are predictive of worse outcomes, indicated by longer overall hospital and intensive care unit stays. Conclusions: Taken together, activin is a novel candidate as a clinical marker and therapeutic target in acute pancreatitis.
Tu1354 SIMULTANEOUS ACTIVATION OF K-RAS AND NRF2 INDUCE PANCREATIC ATROPHY Shin Hamada, Atsushi Masamune, Keiko Taguchi, Masayuki Yamamoto, Tooru Shimosegawa Background: The Keap1-Nrf2 system plays pivotal roles that induce wide variety of cytoprotective genes responsible for cell proliferation and metabolic reprogramming. Aberrant Nrf2 activation is frequently observed in cancer cells, yielding growth and survival advantages in the progression. To clarify the role of Nrf2 in pancreatic carcinogenesis using mutant K-ras and p53 mouse model, we examined if Nrf2 activated by Keap1 deletion facilitates the cancer. Materials and methods: LSL-K-rasG12D, LSL-p53R172H, and Pdx-1-Cre mice were crossed with Keap1fl/fl and/or Nrf2-/- mice to generate mice with genotypes summarized in figure 1. Mice were treated according to the Guidelines for Proper Conduct of Animal Experiments of the Ministry of Education, Culture, Sports, Science and Technology of Japan. These mice were sacrificed at day 90, unless they reached a humane endpoint earlier. Body weight of mice was checked weekly after birth. Serum amylase and glucose were measured at the time of sacrifice. Expression of amylase, insulin, α-smooth muscle actin (α-SMA) and Nqo1 were assessed by immunohistochemistry. Results: Regardless of the presence or absence of p53 loss, mice with K-ras mutant and Keap1 deletion resulted in pancreatic atrophy, which shortened survival time. These mice revealed body weight loss and hypoglycemia, indicating malnutrition. In pancreas with K-ras mutant and Keap1 deletion, Nrf2 activation was evaluated by the elevated expression of typical Nrf2-target gene Nqo1. Amylase positive acinar cells and insulin positive islet cells were reduced in the pancreas of mice with K-ras mutant and Keap1 deletion. Degenerative acinar cells and islet cells were surrounded by α-SMA positive stromal cells, suggesting fibrosis. This phenotype was dependent on Nrf2, because Nrf2 deletion rescued the phenotype in K-ras mutant and Keap1 deletion. Discussion: Pancreas-specific activation of K-ras and Nrf2 caused malnutrition due to loss of pancreatic parenchyma, rather than acceleration of carcinogenesis (figure 2). Nrf2 activation in the context of K-ras mutation might have alternative roles.
AGA Abstracts
Tu1356 SITE-1-PROTEASE MEDIATED UNFOLDED PROTEIN RESPONSE PROTECTS AGAINST PANCREATIC INJURY IN ACUTE PANCREATITIS Kaylene Barrera, Kei Okochi, Albert Stanek, Cathy Mueller, Peiqi Ou, Antonio Alfonso, Chongmin Huan Introduction: Acute pancreatitis (AP) mainly involves acinar cell injury that is initiated when cellular self-protection is overwhelmed by pathogenic dysregulation. Although premature intracellular activation of trypsinogen was widely thought to induce AP, recent studies show that endoplasmic reticulum (ER) stress triggered by multiple AP predisposing factors leads to acinar injury if not salvaged by the unfolded protein response (UPR). Site-1-protease (S1P) is a Golgi membrane-bound UPR mediator that enables activating transcription factor 6 (ATF6) to activate transcription of UPR-target genes via cleavage activation of ATF6. But the self-protective role of S1P-mediated UPR in AP is unknown. Here we study cerulein AP and ethanol-induced acinar abnormalities in S1P deficient mice. Methods: Paired S1Pflox/+ and S1Pflox/flox littermates that express normal level and 70% less pancreatic S1P respectively were studied. Cerulein AP was induced via 7 hourly cerulein (50µg/kg) intraperitoneal injections. Ethanol was administrated by following a binge-like alcohol intake protocol. Pancreatic injury was analyzed by histological and biochemistry studies. Isolated S1Pflox/+ and S1Pflox/flox acinar cells were cultured with ethanol or cerulein for measurement of ER stress-induced S1P mRNA and nuclear ATF6 levels by RT-PCR and Western blot respectively. Results: Cerulein and ethanol treatment increased S1P expression and nuclear ATF6 in acinar cells. Compared to S1Pflox/+ mice, S1Pflox/flox mice had significantly increased serum amylase and lipase, pancreatic acinar injury and inflammatory infiltration in cerulein AP (Figure 1). Consistently, ethanol intake caused higher serum amylase levels and more abnormal pancreatic acinar cells in S1Pflox/flox mice compared to S1Pflox/+ mice (Figure 2). Conclusion: Induced S1P activity in AP protects against acinar cell injury.
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Cerulein induced acute pancreatitis. (L) S1Pflox/+ (R) S1P flox/flox Tu1359 EFFECT OF OCTREOTIDE ON PANCREATIC FIBROSIS OF HIGH-FAT DIET INDUCED OBESITY IN RATS Ting Ye, Rui Liu Objective High-fat diets can induce inflammatory response of pancreas that ultimately lead to pancreatic fibrosis, which is related to activation of pancreatic stellate cells (PSCs). Previous studies have suggested that somatostatin (SST) and its analogs were effective in treating hepatic fibrosis through inhibiting hepatic stellate cells specifically. The aim of this study was to investigate the effects of octreotide on experimental pancreatic fibrosis in obesity rats by high-fat diet (HFD) induced. Methods Sprague-Dawley (SD) male rats were randomly assigned to control, HFD, or octreotide-treated groups. Glucose and insulin tolerance test were determined then calculate the area under the curve (AUC) of the tests. Changes in pancreatic morphology; fasting plasma glucose (FPG) and fasting insulin (FINS); serum triglyceride (TG), total cholesterol (TC), free fatty acid (FFA); pancreatic TG were measured, and Lee's index and homeostatic model assessment (HOMA) index were also calculated. The expression levels of alpha-smooth muscle actin(α-SMA) , desmin, connective tissue growth factor(CTGF), transforming growth factor beta1(TGF-β1),Smad3, Smad7 in pancreas were quantified. Rat pancreatic stellate cells were divided into three groups: (1) control group; (2) palmitate (PA) group; (3)octreotide-treated group, and the expression of α-SMA, CTGF, TGF-β1,Smad3,Smad7 were quantified. Results Compared with the control group, body weight, Lee's index, AUC of ipGTT and ipITT, FPG, FINS, TG, TC and FFA levels, and HOMA index in HFD group were significantly increased. The values of some of these parameters were significantly reduced after octreotide treatment, while Lee's index, TC, TG levels, and AUC were tended to decrease but have no statistical significance. The expression levels of α-SMA, CTGF, TGF-beta1, Smad3 protein and mRNA were significantly higher and Smad7 protein and mRNA level were significantly lower in HFD group than those in the control group. After octreotide treatment, α-SMA, CTGF, TGF-β1, Smad3 protein and mRNA levels were decreased and Smad7 increased. In vitro study, we found that the expression levels of CTGF, TGF-β1, Smad3 protein and mRNA were increased in PA group, and octreotide-treatment down-regulated the expression levels of these molecules. The Smad7 and α-SMA protein and mRNA levels did not show significant changes in PA group or in octreotide-adminstrated group. Conclusion Octreotide can ameliorate pancreatic fibrosis and improve the pancreatic beta-cell function in HFD induced rats, possibly via inhibiting PSC activation and decreasing the pancreatic extracellular matrix (ECM) through TGF-β1/Smad signaling pathway.
Alcohol binge drinking model. (L)S1Pflox/+ (R) S1Pflox/flox
Tu1357 NOVEL SMALL MOLECULE PKD INHIBITORS SUPPRESS NF-ΚB ACTIVATION, INFLAMMATION, NECROSIS AND SEVERITY IN PANCREATITIS Jingzhen Yuan, Meng Geng, Tanya Tan, Grace Tan, Stephen J. Pandol Background and Aims: Acute pancreatitis is characterized by inflammation and necrosis of the parenchymal cells in the pancreas. The pro-inflammatory transcription factor NF-κB plays a critical role in regulation of inflammatory and cell death responses. We previously reported that protein kinase D (PKD) regulates NF-κB activation induced by CCK8 or carbachol in pancreatic acinar cell line AR42J cells. With pharmacological approach, we further showed that NF-κB activation was markedly suppressed by two novel small molecule PKD inhibitors in experimental models of acute pancreatitis, demonstrating that PKD is an important mediator of NF-κB activation in pancreatitis. The aim of the current studies was to continually explore whether suppressing of NF-κB activation by the PKD inhibitors was associated with attenuation of inflammatory responses and severity of the disease in experimental model of pancreatitis. Methods: Sprague-Dawley rats were pre-treated with intraperitoneal (IP) injection of PKD inhibitor CID755673 (CID, 15mg/kg) or CRT0066101 (CRT, 10mg/kg) for 60 min prior to initiation of pancreatic stress with up to 4 hourly IP injections of cerulein (20mg/kg) or saline. Then the animals were sacrificed in 30 min after first and fourth hourly IP injection of cerulein. NF-κB-DNA binding activity in pancreatic tissue nuclear extracts was measured with electrophoretic mobility shift assay. Western blotting was performed to analyze PKD phosphorylation, NF-κB nucleus translocation, and pancreatic protein level of IL-6 with specific antibodies. Pancreatitis parameters were evaluated on H&E stained pancreatic tissue sections. Results: We found that either CID or CRT blocked cerulein-induced PKD autophosphorylation at Ser916 without affecting PKCdependent Ser744/748 phosphorylation in pancreatitis, demonstrating specificity of the PKD inhibitor. PKD inhibition resulted in markedly suppressed NF-κB-DNA binding activity in nuclear extracts and NF-κB nucleus translocation. Importantly, NF-κB inhibition by the PKD inhibitor CID was associated with significantly decreased inflammatory responses and necrosis indicated by reduced pancreatic IL-6 protein, less inflammatory cell infiltration and myeloperoxidase positively stained cells in pancreatic tissue sections, and improved pancreatic necrosis, edema and vacuolization. CONCLUSION: PKD is an important mediator of NF-κB activation in pancreatitis. NF-κB inhibition by specific PKD inhibitor was associated with decreased inflammatory responses and attenuated necrosis and pancreatitis severity in a model of acute pancreatitis. The results suggest that the novel small molecule PKD inhibitors have high potential as therapeutic agents to prevent recurrent pancreatitis through suppressing NF-κB activation.
Tu1360 EFFECTS OF UNSATURATED FREE FATTY ACIDS (UFFA) ON HUMAN ENDOTHELIAL CELLS: IS THERE A THRESHOLD UFFA LEVEL CAUSING CELL TOXICITY AND DEATH IN ACUTE PANCREATITIS? Anna C. Evans, Annette Wilson, Georgios I. Papachristou, David C. Whitcomb BACKGROUND: Hypertriglyceridemic (HTG) acute pancreatitis (AP) is often severe, with up to 30% mortality. While HTG itself is rather benign, release of pancreatic lipase(s) into the circulation during an episode of AP facilitates massive hydrolysis of TG to free fatty acids (FFA). In addition to the pathogenic mechanism of typical AP, release of linoleic acid (LA) and other uFFA causes direct toxicity in animal models. In humans, endothelial injury or stress results in a vascular leak syndrome (VLS), linking systemic inflammation with a multi-organ dysfunction syndrome. It is not known if uFFA affect human microvascular endothelial cells, and if any pathologic effect is concentration-dependent. Since two pathogenic processes are possible in HTG-AP (cytokine storm and lipotoxicity), it is important distinguish the two processes. Knowing the effect of uFFA on human endothelial cells is critical to solving this problem. HYPOTHESIS: We hypothesize that Linoleic Acid(LA) causes increased cell death of in human intestinal microvascular endothelial cells (HiMECs), thereby causing the disruption of endothelial barrier integrity, eventually resulting in vascular leak syndrome independent of cytokines. METHODS: Human intestinal microvascular epithelial cells were isolated from surgical margin specimens using CD31 marker beads. They were passaged in vitro and passages 5-15 were used for experimentation. Linoleic Acid (18:2) was dissolved in DMSO to create solutions of increasing concentration and applied directly to cells for 24h. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) was tested using colorimetric assay. LDH (Lactate Dehydrogenase) was tested also using colorimetric assay. One-way ANOVA and post-hoc Tukey tests were used to determine significant differences. RESULTS: Endothelial cells treated with increasingly concentrated solutions of Linoleic Acid (18:2) have decreasing viability (Fig 1). Absorbance was measured as Optical Density (OD) and was read by a BioRad Microplate Reader with 570nM filter. Bars show average reading of optical density (n=18) with standard deviation. Significant differences (p<0.05) are seen between controls and concentrations of 1.25mM, 2.5mM, and 5.0mM Linoleic Acid. Increased levels of LDH were also found with increased concentrations of Linoleic Acid. Significant differences were found between cells treated w 2.5mM Linoleic Acid, 5.0mM Linoleic Acid, and controls. CONCLUSIONS: uFFA such as LA causes direct cell toxicity in human microvascular endothelial cells at concentrations at, or above 1.25 mM. Clinical studies in humans are needed to correlate uFFA levels and evidence of VLA and lipotoxicity, followed by trials to rapidly clear FFA when the concentration exceed the threshold for lipotoxicity.
Tu1358 LIPOTOXICITY MAY RESULT IN INFLAMMATORY CELL DEATH AND ASSOCIATED IMMUNE PARALYSIS DURING SEVERE ACUTE PANCREATITIS (SAP) Bara El Kurdi, Jordan R. Yaron, Shehryar Masood, Pawan Noel, Krutika Patel, Biswajit Khatua, Cristiane Oliveira, Vijay P. Singh Background: Patients with SAP can develop the Compensatory Anti-inflammatory Response syndrome (CARS), associated with opportunistic infections. Additionally, a White blood cell (WBC) count < 4000/mm3 is part of the criteria for Systemic Inflammatory Response Syndrome (SIRS) which can develop in SAP. This study was designed to understand these phenomena. Methods: The Mayo clinic autopsy database was searched for patients with who died with necrotizing pancreatitis (NP; n=8) or unrelated causes (controls). Age, gender, BMI were recorded and spleens of these were stained for apoptotic cells (TUNEL) and quantified as % positive nuclei. Additionally, SAP was induced in Wistar rats (250gm) by administration of caerulein (CER; 50mcg/kg BID) and glyceryl trioleate (GTO) alone or along with the lipase inhibitor orlistat (GTOO) as described previously [PMID 25500204]. WBC counts, fatty acids were quantified in the blood of these rats and TUNEL staining was done on their spleens and quantified as number of positive cells per white pulp pixel area. A p of <0.05 was regarded as significant. Results: The mean BMI of patients with NP was 45.6 kg/m2 vs. 26.1 kg/m2 in controls. NP was associated with an increase in TUNEL positive cells in the spleen (1% vs. 10%, p=0.0117) including the white pulp (1% vs. 10%, p= 0.0155). The CER+GTO group had 100% mortality (vs. 0% in all other groups). This was
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associated with a 6.4 fold increase in TUNEL positive cells in the white pulp of the spleens of rats in the CER+GTO group vs. controls (p<0.0001) and a decrease in total WBC count from (12.2±1.2×103/mm3 in control to 3.5±0.5×103/mm3, p<0.02), along with an increase in circulating free fatty acids (539±116µM vs 252±91µM in controls p<0.003). These changes were prevented in the CER+GTOO group, which was not significantly different from controls. Conclusion: CARS can result from increased inflammatory cell death during severe illnesses such as necrotizing pancreatitis. This may be seen as a reduction in circulating WBC counts and an increase in apoptotic inflammatory cells- as we note in the spleens during SAP. Targeting lipotoxicity form the fatty acids released from visceral fat lipolysis may reduce CARS during SAP and the immune paralysis associated with it.