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Protective effects by overexpression of Hsp27 against caerulein-induced acute pancreatitis
similar serum amylase (2517 vs. 3017u, p=0.22), pancreatic edema (71.5 vs.72.5% p=0.34), trypsin/DNA content (0.13 vs 0.09, p = 0.32) and acinar cell morphology. After ] 2 hours of pancreatitis, the wild types had a serum amylase of 21470u, pancreatic edema of 78.2%, and necrosis of 18.5% AII of these were markedly increased in the PAR-2 K/O /serum amylase-98135u, pancreatic edema-84.9% and necrosis-32.6% (all wath p<0.05 compared to WT)). Trypsmogen activation, I-KB degradation, NF-KB and AP-1 activation were similar in both groups at 30 minutes. However at 6 hours, while the WT had sustained [-KBn and ~3 degradation, both reappeared in the PAR-2 K/O. AP-1 activation was also markedly less in the PAR-2 K/O at 6 hours. However, the PAR-2 K/O had markedly more Irypsin/DNA activuy at 6 hours as compared to WT (08i vs. 0.28 p<0.05). CONCLUSIONS: Genetic ddetion of PAR-2 worsens the severity of pancreatitis independent of AP- 1 activation or sustained I-KB degradation known to occur ni pancreatins. This may be linked to the increased trypsin activity observed in the PAR-2 K/O. The exacerbation of the pancreatic injury in the absence of PAR-2 indicates that PAR-2 plays an anti-inflammatory role in pancreatitis. This is in contrast to its pro-inflammatory role reported in other systems
738
Pancreatitis-Associated Protein is Protective Against Acute Pancreatiris Emad KandiI, Hong Zhang, Gabriel Levi, Yinyao Lin, Codcin Nemes, Domenico Viterbo. Gordon Callender, Victor Ocasio, Michael Zenilman Background: Pancreatitis-associated protein (PAP) is expressed in acinar cells following pancreatitis, but its functional role is not clear. The presence o| at least three isoforms has precluded gene knockout experiments. To determine if PAP expression is protective in pancreatitis, we administered antisense oligonucleotides (ASO-PAP) to all three subtypes (PAP I, II, and Ill). Methods: Pancreatitis was induced in rats by retrograde infusion of 4% sodium taurocholate into the pancreatic duct. Pretreatmem with ASO-PAP, scrambled oligonudeotides or saline controls were given via pancreatic subcapsular injection 24 hours prior to pancreatitis induction (n = 8 for each group). Pancreata, serum, and splenic lymphocytes were harvested 24 hours after the induction. Severity of pancreatitis was based on serum amylase, C-reactive protein (CRP), pancreatic wet weigh, cytokine gene expression profile of lymphoeytes and bistopatholog)cal score. Results: RT-PCR of pancreta after induction of pancreatitis showed partial or complete inhibition of PAP genes in 83% of ASO-PAP treated-animals vs~ 0% of controls (Figure). Significant changes were noted in all parameters after nihihition of PAP expression (Table). Histopathologic analysis revealed that after inhibition of PAP inflammatory changes correlated dosdy with serum amylase levels (r = .6, P < ,001) and also with serum CRP (r = .5, P < .05). Similarly, pancreatic necrosis con'elated closely with serum amylase levels (r = .6, P < .001) and also with the wet weight (r = 4, P = .05). Condnsions: There was significant worsening of pancreatitis after intrapancreatic ASO-PAP administration compared to controls inhibition of the PAP genes exacerbated parameters associated with pancreatitis such as inflammation, CRP elevation, and pancreatic necrosis. Results indicate that PAP induction during injury mediates significant protection against pancreatitis and underscores the role of PAP as an autocrine protector of pancreas from injury.
736
Iransgenic Expression of Pancreatic Secretory Trypsin Inhibitor-I (PSTbl) in Mice Protects Against Caerulein-lnduced Chronic Pancreatitis and Reduces Pancreatic Fibrosis Jaimie D. Nathan, Joelle Romac, Ruth Y Peng, Michael Peyton, Don C Rockey, Raymond J. MacDonald, Rodger A. hddle BACKGROUND: We recently demonstrated that transgenic expression of rat pancreatic secretory trypsin inhibitor-I (PSTI-i) in mice ameliorates secretagogue-induced acute pancreatitis. In the current study, we tested whether the seventy of chronic pancrearitis and accompanying pancreatic fibrosis is reduced in mice that express PSTM. METHODS: PSTi[ expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the mouse elastase promoter/enhancer. Among 10 organs examined, PSTII was detected by Western analysis only in the pancreas Chronic pancreatitis was achieved by seven hourly intrapentoneal injections of the pancreauc secretagogne caerulein (50 lig/ kg) semiweekly for ten weeks. Control animals received injections of vehicle After ten weeks, the severity of chronic pancreatitis was assessed by histological grading of inflammatory infiltrate, atrophy, and fibrosis, and by quantitative morphometrical analysis of pancreatic cdlagen content (using Sirius Red). RESULTS: Caerulein administration to non-transgenic miceproduced histological evidence of inflammatory infiltrate, glandular atrophy, and parenehymaI fibrosis, and significantly increased collagen staining in pancreatic sections. In PSTII-expressing mice that received caerulein, there were significant reductions in inflammatory infihrate (0.63 -+ 0.13 vs. 1.75 + 048; p < 005; n = 4) and fibrosis (1.88 -.+ 0.38 vs. 425 _+ 0.48; p < 0.01). The total histological score was reduced in mice expressing PSTI1 from 8.63 _+ 0.85 to 4.38 _+ 0.38 (p < 0.01) Quantitative morphometrical analysis demonstrated that PSTI-I-expressing mtce treated with caerulein (5.06 -~ 2.13 96 area), had significantly less collagen than non-transgenic mice treated with caerulein (20.81 _+ 2.93 96 area; p < 0001). CONCLUSIONS: The severity of chronic pancreatitis and pancreatic fibrosis induced by long-term repeated caerulein injections is significantly reduced m mice expressing rat pancreatic secretory' trypsin inhibitor-I. We propose that endogenous pancreatic trypsin inhibitors play a protective role in the pancreatic parenchymal response to repeated injurious events.
TaMe, PancreatJtl$ pmameters after treal~ent of ASO.PAP, scrambled ollgonucleotkle or saline control.
Mean _+SEM
Amylase (U/L)
CRP (mg/dL)
6938.0 ~ 1066.7"
3.4 0,1"
WatWt (% of body
Inflammation (score)
% Of Necroais
% Of Survival
0.0'
1,8 ! 0.4*
666.P/o*
75%
V; og.o,
,.o o.1 14.3%
100%
wt) ASO..PAP Scrambled
1970.0 ~
=,
2613.7,2.
351.,
2.0 0.1 2.4 0.2
0.7
1.1 0.'i
* P < .05 compared to control by ANOVA analysis.
Scrambled
PAP
I
PAP
III
ASCAP
737
Protective Effects by Overexpression of Hsp27 Against Caerulein-induced Acute Pancreatitis Constanze Kuhisch, Matt J. Dimagno, Michael J. Welsh, Steve A. Ernst, Susanne Arbogast, Burkhard Goeke, Andreas C C. Wagner, John A. Williams, Claus Schaefer Introduction: Hsp27is involved in the regulation of the actin cytosketeton and overexpmssion in fibroblasts confers resistance against stress. In addition, the protective effects of Hsp27 are dependent on the phosphorylation of Hsp27. Redistribution of pancreatic acinar actin cytoskeleton occurs during supramaximal stimulation with CCK m vivo and in vitro and is thought to reflect cell injury. We hypothesized that overexpression of HSP27 in the pancreas would be protective against acute pancreatitis. Material and Methods: Transgenic mice overexpressing human Hsp27 (wt-Hsp27) or mutated forms of Hsp27 in the pancreas were developed using a construct containing Hsp27 under control of the CMV promoter. Hsp27 was mutated at the three phosporylation sites to alanine which can not be phosphorylated (JA-Hsp27) or to aspartic acid (3D-Hsp27) which mimics the phosphorylated form of HSP27.Protein expression in the pancreas was demonstrated by western blotting. Pancreatitis wasinduced by hourly i.p. injection of 50 p.g/kg/bw caerulein over 6 or 12 hours. Pancreatitis was determined by measunng serum amylase and lipase, trypsinogen activation, pancreas water content and assessing morphology by H&E staining and with rhodamine-phalloidin for the actin cytoskdeton. Resutts: Western blotting revealed a strong pancreatic expression of human Hsp27 and localisation by immunohistochemistry showed expression in exoerine acini Mice with overexpression of wt-Hsp27 and 3D-Hsp27 showed significantly less severe pancreatitis with reduced serum amylase and lipase, reduced water-content and trypsin activityafter 6 and 12 hours compared to non-transgenic mice or 3A-Hsp27 mice. Pancreatic histology showed only minimal changes 6 hrs. after caerulem-injections, whereas major changes were observed after 12 hours in non-transgenic and 3A-Hsp27 mice and included lalracellular vacuoles, single cell necrosis and edema. Wt-Hsp27 and 3D-Hsp27 mice showed significantless severe changes; the actin cytoskeleton was better preserved in these animals. Conclusion: The overexpression of wt-Hsp27 or 3D-Hsp27 but not the non-phosphorylatable mutant 3A-Hsp27 is protective against caeruleinqnduced pancreatitis with inhibition of trypsin activation and protection of the actin cytoskelaton. This is, to our knowledge, the first report, that overexpression of a single heat shock protein leads to significant protective dfects in vivo in the pancreas
739 Deletion of the Calcium Binding Protein $100A9 (MRP14) Reduces Experimental Pancreatitis Verena Hlonschek, Juergen Schnekenburger, Wolfgang Nacken, Wolfram Domschke, Clemens Sorg, Markus M. Lurch $100A9 (MRP14) is a calcium binding protein expressed by myeloid cells and increased serum levels of MRPI4 have been observed in various inflammatory and autoimmune disorders. We have studied the role of MRP14 in the course of acute experimental pancreatitis. MRP14 knock out mice were generated by target deletion of the MRP14 gene and these animals expressed no visible phenotype. Acute pancreatitis was induced by 7 i.p Caerulein injections (50g/kg) in MRP14 kock out or C57BL/6 wild type mice. The pancreas was either homogenized for protein analysis, or fixed for histology and immunocytochemistry. We further determined trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities and tissue myeloperoxidase (MPO) activity. During acute pancreatitis MRPI4 was only detected in infiltrating pancreatic lenkocytes of wild type animals. In MRP14 knock out mice Caerulein pancreatitis was associated with significantly lower serum amylase and lipase activities, reduced intrapancreatic TAP levels and reduced MPO activity in the lungs and pancreas. The reduced MPO activity in the lungs and the pancreas indicate that MRP14 is
A-95
AGA Abstracts
738
Pancreatitis-Associated Protein is Protective Against Acute Pancreatiris Emad KandiI, Hong Zhang, Gabriel Levi, Yinyao Lin, Codcin Nemes, Domenico Viterbo. Gordon Callender, Victor Ocasio, Michael Zenilman Background: Pancreatitis-associated protein (PAP) is expressed in acinar cells following pancreatitis, but its functional role is not clear. The presence o| at least three isoforms has precluded gene knockout experiments. To determine if PAP expression is protective in pancreatitis, we administered antisense oligonucleotides (ASO-PAP) to all three subtypes (PAP I, II, and Ill). Methods: Pancreatitis was induced in rats by retrograde infusion of 4% sodium taurocholate into the pancreatic duct. Pretreatmem with ASO-PAP, scrambled oligonudeotides or saline controls were given via pancreatic subcapsular injection 24 hours prior to pancreatitis induction (n = 8 for each group). Pancreata, serum, and splenic lymphocytes were harvested 24 hours after the induction. Severity of pancreatitis was based on serum amylase, C-reactive protein (CRP), pancreatic wet weigh, cytokine gene expression profile of lymphoeytes and bistopatholog)cal score. Results: RT-PCR of pancreta after induction of pancreatitis showed partial or complete inhibition of PAP genes in 83% of ASO-PAP treated-animals vs~ 0% of controls (Figure). Significant changes were noted in all parameters after nihihition of PAP expression (Table). Histopathologic analysis revealed that after inhibition of PAP inflammatory changes correlated dosdy with serum amylase levels (r = .6, P < ,001) and also with serum CRP (r = .5, P < .05). Similarly, pancreatic necrosis con'elated closely with serum amylase levels (r = .6, P < .001) and also with the wet weight (r = 4, P = .05). Condnsions: There was significant worsening of pancreatitis after intrapancreatic ASO-PAP administration compared to controls inhibition of the PAP genes exacerbated parameters associated with pancreatitis such as inflammation, CRP elevation, and pancreatic necrosis. Results indicate that PAP induction during injury mediates significant protection against pancreatitis and underscores the role of PAP as an autocrine protector of pancreas from injury.
736
Iransgenic Expression of Pancreatic Secretory Trypsin Inhibitor-I (PSTbl) in Mice Protects Against Caerulein-lnduced Chronic Pancreatitis and Reduces Pancreatic Fibrosis Jaimie D. Nathan, Joelle Romac, Ruth Y Peng, Michael Peyton, Don C Rockey, Raymond J. MacDonald, Rodger A. hddle BACKGROUND: We recently demonstrated that transgenic expression of rat pancreatic secretory trypsin inhibitor-I (PSTI-i) in mice ameliorates secretagogue-induced acute pancreatitis. In the current study, we tested whether the seventy of chronic pancrearitis and accompanying pancreatic fibrosis is reduced in mice that express PSTM. METHODS: PSTi[ expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the mouse elastase promoter/enhancer. Among 10 organs examined, PSTII was detected by Western analysis only in the pancreas Chronic pancreatitis was achieved by seven hourly intrapentoneal injections of the pancreauc secretagogne caerulein (50 lig/ kg) semiweekly for ten weeks. Control animals received injections of vehicle After ten weeks, the severity of chronic pancreatitis was assessed by histological grading of inflammatory infiltrate, atrophy, and fibrosis, and by quantitative morphometrical analysis of pancreatic cdlagen content (using Sirius Red). RESULTS: Caerulein administration to non-transgenic miceproduced histological evidence of inflammatory infiltrate, glandular atrophy, and parenehymaI fibrosis, and significantly increased collagen staining in pancreatic sections. In PSTII-expressing mice that received caerulein, there were significant reductions in inflammatory infihrate (0.63 -+ 0.13 vs. 1.75 + 048; p < 005; n = 4) and fibrosis (1.88 -.+ 0.38 vs. 425 _+ 0.48; p < 0.01). The total histological score was reduced in mice expressing PSTI1 from 8.63 _+ 0.85 to 4.38 _+ 0.38 (p < 0.01) Quantitative morphometrical analysis demonstrated that PSTI-I-expressing mtce treated with caerulein (5.06 -~ 2.13 96 area), had significantly less collagen than non-transgenic mice treated with caerulein (20.81 _+ 2.93 96 area; p < 0001). CONCLUSIONS: The severity of chronic pancreatitis and pancreatic fibrosis induced by long-term repeated caerulein injections is significantly reduced m mice expressing rat pancreatic secretory' trypsin inhibitor-I. We propose that endogenous pancreatic trypsin inhibitors play a protective role in the pancreatic parenchymal response to repeated injurious events.
TaMe, PancreatJtl$ pmameters after treal~ent of ASO.PAP, scrambled ollgonucleotkle or saline control.
Mean _+SEM
Amylase (U/L)
CRP (mg/dL)
6938.0 ~ 1066.7"
3.4 0,1"
WatWt (% of body
Inflammation (score)
% Of Necroais
% Of Survival
0.0'
1,8 ! 0.4*
666.P/o*
75%
V; og.o,
,.o o.1 14.3%
100%
wt) ASO..PAP Scrambled
1970.0 ~
=,
2613.7,2.
351.,
2.0 0.1 2.4 0.2
0.7
1.1 0.'i
* P < .05 compared to control by ANOVA analysis.
Scrambled
PAP
I
PAP
III
ASCAP
737
Protective Effects by Overexpression of Hsp27 Against Caerulein-induced Acute Pancreatitis Constanze Kuhisch, Matt J. Dimagno, Michael J. Welsh, Steve A. Ernst, Susanne Arbogast, Burkhard Goeke, Andreas C C. Wagner, John A. Williams, Claus Schaefer Introduction: Hsp27is involved in the regulation of the actin cytosketeton and overexpmssion in fibroblasts confers resistance against stress. In addition, the protective effects of Hsp27 are dependent on the phosphorylation of Hsp27. Redistribution of pancreatic acinar actin cytoskeleton occurs during supramaximal stimulation with CCK m vivo and in vitro and is thought to reflect cell injury. We hypothesized that overexpression of HSP27 in the pancreas would be protective against acute pancreatitis. Material and Methods: Transgenic mice overexpressing human Hsp27 (wt-Hsp27) or mutated forms of Hsp27 in the pancreas were developed using a construct containing Hsp27 under control of the CMV promoter. Hsp27 was mutated at the three phosporylation sites to alanine which can not be phosphorylated (JA-Hsp27) or to aspartic acid (3D-Hsp27) which mimics the phosphorylated form of HSP27.Protein expression in the pancreas was demonstrated by western blotting. Pancreatitis wasinduced by hourly i.p. injection of 50 p.g/kg/bw caerulein over 6 or 12 hours. Pancreatitis was determined by measunng serum amylase and lipase, trypsinogen activation, pancreas water content and assessing morphology by H&E staining and with rhodamine-phalloidin for the actin cytoskdeton. Resutts: Western blotting revealed a strong pancreatic expression of human Hsp27 and localisation by immunohistochemistry showed expression in exoerine acini Mice with overexpression of wt-Hsp27 and 3D-Hsp27 showed significantly less severe pancreatitis with reduced serum amylase and lipase, reduced water-content and trypsin activityafter 6 and 12 hours compared to non-transgenic mice or 3A-Hsp27 mice. Pancreatic histology showed only minimal changes 6 hrs. after caerulem-injections, whereas major changes were observed after 12 hours in non-transgenic and 3A-Hsp27 mice and included lalracellular vacuoles, single cell necrosis and edema. Wt-Hsp27 and 3D-Hsp27 mice showed significantless severe changes; the actin cytoskeleton was better preserved in these animals. Conclusion: The overexpression of wt-Hsp27 or 3D-Hsp27 but not the non-phosphorylatable mutant 3A-Hsp27 is protective against caeruleinqnduced pancreatitis with inhibition of trypsin activation and protection of the actin cytoskelaton. This is, to our knowledge, the first report, that overexpression of a single heat shock protein leads to significant protective dfects in vivo in the pancreas
739 Deletion of the Calcium Binding Protein $100A9 (MRP14) Reduces Experimental Pancreatitis Verena Hlonschek, Juergen Schnekenburger, Wolfgang Nacken, Wolfram Domschke, Clemens Sorg, Markus M. Lurch $100A9 (MRP14) is a calcium binding protein expressed by myeloid cells and increased serum levels of MRPI4 have been observed in various inflammatory and autoimmune disorders. We have studied the role of MRP14 in the course of acute experimental pancreatitis. MRP14 knock out mice were generated by target deletion of the MRP14 gene and these animals expressed no visible phenotype. Acute pancreatitis was induced by 7 i.p Caerulein injections (50g/kg) in MRP14 kock out or C57BL/6 wild type mice. The pancreas was either homogenized for protein analysis, or fixed for histology and immunocytochemistry. We further determined trypsinogen activation peptide (TAP) levels, serum amylase and lipase activities and tissue myeloperoxidase (MPO) activity. During acute pancreatitis MRPI4 was only detected in infiltrating pancreatic lenkocytes of wild type animals. In MRP14 knock out mice Caerulein pancreatitis was associated with significantly lower serum amylase and lipase activities, reduced intrapancreatic TAP levels and reduced MPO activity in the lungs and pancreas. The reduced MPO activity in the lungs and the pancreas indicate that MRP14 is
A-95
AGA Abstracts