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Activin in Acute Pancreatitis: Potential Risk-Stratifying Marker and Novel Therapeutic Target
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.
S-894
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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