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Akt1 regulates the development of inflammation and tissue regeneration during acute pancreatitis
Abstracts / Pancreatology 16 (2016) S1eS130
signaling in favor of oncogenic properties. Importantly, loss of Smad4 favors complex formation of Smad3 with oncogenic partner proteins. We previously identified the oncogenic Nuclear Factor of activated T cells (NFATc1) transcription factor as a prominent target of oncogenic TGFbsignaling. However, the mechanism of TGFb-controlled NFATc1-dependent transcription in Smad4-deficient PDAC remains elusive. Aims: To characterize NFATc1/Smad3 complex formation, regulation and function in Smad4-deficient pancreatic carcinogenesis and PDAC progression. Materials & methods: NFATc1/Smad3 assembled chromatin complexes are characterized in vitro and in vivo in PDAC cell lines with CRISPR/ Cas9-mediated Smad4 knock out and in Smad4-deficient genetically engineered mice. NFATc1/Smad3 regulated gene signatures are analyzed by genome-wide DNA binding and expression studies. Results: Co-immunoprecipitation experiments revealed Smad4dependent NFATc1/Smad3 transcription complex formation. Reexpression of Smad4 resulted in NFATc1/Smad3 complex disruption, induced apoptosis and decreased cell cycle progression. While TGFb-signaling is not crucial for the formation of the transcription complex, it enhances its stabilization and has an impact on its transcriptional activity. Conclusion: Here we identify nuclear NFATc1/Smad3 complex formation as a key component of oncogenic TGFb-signaling in Smad4-deficient PDAC. Targeting this complex might be a beneficial approach in a subgroup of patients with Smad4-deficient pancreatic cancer.
Abstract ID: 1447. Pancreatic ductal adenocarcinoma (PDAC)-derived exosomes enrich the microenvironment in Myeloid Derived Suppressor Cells (MDSCs) in a Smad4-dependent manner through a new calcium related axis Elisa Gnatta 1, Sara Furlanello 1, Ada Aita 1, Paola Fogar 1, Chiara Frasson 2, Dania Bozzato 1, Andrea Padoan 1, Basso Giuseppe 2, Mario Plebani 1, Daniela Basso 1 1 2
Department of Medicine-DIMED, University of Padova, Italy Department of Woman and Child Health, University of Padova, Italy
Introduction: Genetic aberrations (including Smad4 inactivation) and immunoevasion concur in worsening PDAC prognosis. Calcium signalling plays a relevant role in the elaboration of the adaptive immune response against cancer. Aims: To verify whether PDAC affects immune cells in a Smad4dependent manner and whether tumor-derived exosomes play a part by altering intracellular calcium ([Ca2+]i) fluxes. Materials & methods: 1%FCS-Conditioned media (CM) from two pancreatic cancer cell lines BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) were used. Peripheral blood mononuclear cells (PBMCs) from 8 blood donors were cultured (4 days) in unfractioned control medium, BxPC3/CM and BxPC3-Smad4+/CM, and in Exosomesenriched (pellet), or Exosomes-free (supernatant) media (ultracentrifuCD8+, CD4+CD25+ T-cells, Dendritic gation protocol). CD4+, and two MDSCs subsets cells (CD11b+CD14+DR-¼DCs) (CD11b+CD14-DR+¼mMDSCs; CD11b+CD14-DR-¼gMDSCs) were FACS analyzed. [Ca2+]i fluxes were studied with Fluo-4 stain and recorded by a fluorescent microscope in PBMCs treated in the same conditions. Results: Unfractioned BxPC3-Smad4+/CM significantly induced CD4+CD25+ cells (p¼0.003) while dampening CD8+ cells (p¼0.007) whit respect to control. Both unfractioned PDAC CM significantly dampen DCs (p¼0.009) and gMDSCs (p¼0.013), while they induce mMDSCs (p¼0.006) but only BxPC3-derived exosomes mediate the expansion of both MDSCs populations. Unfractioned BxPC3/CM caused a significant increase in the number of PBMCs with [Ca2+]i peaks (p¼0.009) with respect to control and this was confirmed in Exosomes-enriched CM (p¼ 0.007). After FACS sorted myeloid populations, DCs show a static behaviour while mMDSCs present regular [Ca2+]i peaks. Conclusion: PDAC-derived exosomes from Smad4 expressing, but mainly from Smad4 non-expressing cells, through the increase in [Ca2+]i fluxes, create an immunosuppressive myeloid cells background.
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Abstract ID: 1449, Oral-10. Inhibition of Class I HDAC ameliorates acute and chronic pancreatitis by reducing leukocyte recruitment, acinar-to-ductal metaplasia and fibrosis Marta Bombardo, Enrica Saponara, Ermanno Malagola, Rong Chen, Rolf Graf, Sabrina Sonda Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: Epigenetic mechanisms regulated by the activity of histone deacetylases (HDACs) contribute to the pathology of many inflammatory diseases. Aims: In this study we analyzed whether HDACs are activated during acute (AP) and chronic pancreatitis (CP) and whether pharmacological inhibition of HDACs has a beneficial effect during these diseases. Materials & methods: AP and CP were induced by cerulein injections in wild type C57BL/6 mice. The expression and activity of HDACs and the effects of class I HDAC inhibition with the selective inhibitor MS-275 were evaluated using biochemical, qRT-PCR and imaging techniques. In vitro effects of MS-275 were evaluated using 3D cultures of primary acinar cells and macrophage cell line RAW 264.7. Results: Induction of pancreatitis resulted in increased expression of class I HDACs, increased nuclear HDAC activity and decreased acetylation of nuclear proteins. Inhibitions of class I HDAC with MS-275 significantly reduced pancreatic inflammation both in AP and CP by limiting the migration of neutrophils and macrophages and directly affect macrophage activation. In addition, MS-275 treatment limited the severity of acinar cell damage and prevented the formation of acinar-to-ductal metaplasia, both in vitro and in vivo. Finally, MS-275 reduced the development of fibrosis by down-regulating TGF-b and EGFR signaling in the pancreas. Conclusion: Our investigation demonstrates that class I HDACs play a crucial role in acute and chronic pancreatitis, promoting the development of inflammatory response, metaplastic lesions and tissue fibrosis. In addition, our results show that inhibition of class I HDACs could be a useful treatment for this severe disease.
Abstract ID: 1450. Akt1 regulates the development of inflammation and tissue regeneration during acute pancreatitis Rong Chen 1, Gianluca Mosca 1, Maren Dietrich 2, Enrica Saponara 1, Kamile Grabliauskaite 1, Richard A. Zuellig 2, Oliver Tschopp 2, Rolf Graf 1, Sabrina Sonda 1 1
University Hospital Zurich, Department of Visceral & Transplantation Surgery, Switzerland 2 University Hospital Zurich, Division of Endocrinology, Diabetes and Clinical Nutrition, Switzerland
Introduction: The serine/threonine kinase Akt/PKB plays a key role in the conserved phosphoinositide 3-kinase signaling pathway. In mammals, activation of Akt is implicated in the regulation of divergent cellular processes, including proliferation, survival and cell size. Recently, Akt has also been implicated in the regulation of inflammation through activation of the NF-kB pathway. Three Akt isoforms (Akt1, Akt2, and Akt3) are found in mammals and, despite being highly related and sharing the same structural organization, they show non-redundant physiological functions. Aims: In this study we investigated whether the Akt1 isoform plays a critical role during the development of acute pancreatitis (AP). Materials & methods: AP was induced by repetitive injections of cerulein in wild type (WT) and Akt1-/- mice. The onset and progression of the disease were evaluated in the two mouse strains by serum enzyme quantification, immunohistochemistry and qRT-PCR analyses. Results: Following AP induction, WT and Akt1-/- mice showed comparable initial damage of acinar cells. However, during disease progression, Akt1-/- mice displayed reduced infiltration of inflammatory cells in the
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Abstracts / Pancreatology 16 (2016) S1eS130
pancreas. In addition, lack of Akt1 resulted in reduced acinar cell proliferation and de-differentiation into acinar-to-ductal metaplasia. Molecular analyses revealed a down-regulation of TGF-b signaling in Akt1-/- mice. Conclusion: Our results revealed that Akt1 signaling does not mediate the initial acinar cell damage observed at the onset of AP. However, Akt1 signaling promotes both the inflammatory response and the regeneration of the pancreatic tissue. These findings provide novel insights into the molecular pathways governing pancreatic inflammation and tissue regeneration, with potential therapeutic implications.
Abstract ID: 1451. 1-Deoxy-sphingolipids, novel biomarkers of diabetes, are cytotoxic for exocrine pancreatic cells Rong Chen 1, Thorsten Hornemann 2, Wei Yu 2, Simone Camargo 3, Rolf Graf 1, Sabrina Sonda 1 1 University Hospital Zurich, Department of Visceral & Transplantation Surgery, Switzerland 2 University Hospital Zurich, Institute for Clinical Chemistry, Switzerland 3 University of Zurich, Institute of Physiology, Switzerland
Introduction: Exocrine pancreatic insufficiency and dysfunctions are frequently associated with diabetes mellitus (DM), which also constitutes a risk factor to develop pancreatitis. We recently discovered that deoxysphingolipids (1-deoxySLs) increase during DM and are cytotoxic for beta cells. Aims: Knowing the anatomical and functional relationships between endocrine and exocrine pancreatic tissues, we hypothesize that elevated 1deoxySLs levels observed in DM directly damage the exocrine compartment, thus increasing its predisposition to develop (exocrine) pancreatic diseases. Materials & methods: DM was induced with streptozotocin injections in C57BL/6 mice and Wistar rats. Reduction of 1-deoxySL synthesis was obtained by oral L-serine supplementation. Disease severity was evaluated with biochemical and immunohistochemical methods. Molecular mechanisms of 1-deoxySL-dependent toxicity were evaluated in vitro in pancreatic acinar cells and primary pancreatic fibroblasts. Results: DM induction resulted in increased 1-deoxySL levels but also atrophy and fibrosis of pancreatic parenchima. Reduction of 1-deoxySL synthesis by oral L-serine supplementation ameliorated the damage of the exocrine pancreatic tissue, without restoring insulin production in beta cells. This suggests that elevated 1-deoxySLs rather than insulin deficiency contribute to the exocrine damage in DM. In vitro studies showed that treatment with 1-deoxysphinganine at low micromolar concentration reduced replication and promoted cytotoxicity in pancreatic acinar cells and fibroblasts. Moreover, 1-deoxySL-mediated cytotoxicity was associated with ER stress and ROS production. Conclusion: Our work revealed that 1-deoxySLs are cytotoxic for exocrine pancreatic cells, suggesting a role for these lipids in the exocrine dysfunctions observed following DM. Oral L-serine supplementation could be an option for ameliorating exocrine pancreatic diseases in diabetic patients.
Abstract ID: 1452, Oral-15. The analgesic effect of the mitochondria targeted antioxidant SkQ in experimental pancreatitis Jan D'Haese 1, Maximilian Weniger 1, Leonard Reinelt 1, Jens Neumann 2, Lesca Holdt 3, Matthias Ilmer 1, Bernhard Renz 1, Werner Hartwig 1, Jens Werner 1, Alexandr Bazhin 1 1 LMU Munich, Department of General, Visceral, and Transplantation Surgery, Germany
2 3
LMU Munich, Institute of Pathology, Germany LMU Munich, Institute of Laboratory Medicine, Germany
Introduction: Chronic pancreatitis is one of the main risk factors for pancreatic cancer. In acute and chronic pancreatitis, oxidative stress is thought to play a key role. In this respect, the recently described mitochondria-targeted antioxidant SkQ effectively scavenges reactive oxygen species at nanomolar concentrations. Aims: Therefore, we aimed to characterize the influence of SkQ on tissue injury and pain in acute and chronic pancreatitis. Materials & methods: Both acute and chronic pancreatitis were induced in C57BL/6 mice by intraperitoneal cerulein injections and treatment with SkQ was carried out by peroral applications. Hyperalgesia was assessed by behavioral observation and measurement of abdominal mechanical sensitivity. Blood serum and pancreatic tissue were harvested for analysis of lipase and histology. Results: SkQ did not influence pain, serological or histological parameters of tissue injury in acute pancreatitis. In chronic pancreatitis, a highly significant reduction of pain-related behavior (p < 0.0001) was evident, but histological grading revealed increased tissue injury in SkQtreated animals (p ¼ 0.03). Conclusion: After SkQ-treatment, tissue injury is not ameliorated in acute pancreatitis and increased in chronic pancreatitis. However, we postulate a modest analgesic effect in chronic pancreatitis. Further studies will need to further elucidate the risks and benefits of mitochondria-targeted antioxidants as an analgesic.
Abstract ID: 1453. Non-tumour cell autonomous effects of antiangiogenic multikinase inhibitor regorafenib on infiltrating macrophages in pancreatic neuroendocrine tumours Maren Egidi, Heidi Griesmann, Patrick Michl, Sebastian Krug Department of Gastroenterology and Hepatology, University Hospital, Martin-Luther-University, Halle Saale, Germany Introduction: Pancreatic neuroendocrine tumours (PNETs) represent a distinct hypervascular tumour entity with marked response rates to treatment strategies with antiangiogenic multikinase inhibitors. However, acquired resistance frequently develops and limits the long-term benefit of antiangiogenic approaches. Infiltration of immune cells including tumourassociated macrophages (TAMs) has been associated with tumour progression and increased angiogenesis. Aims: Evaluation of the functional impact of antiangiogenic multikinase inhibitors such as regorafenib on infiltrating macrophages in pancreatic neuroendocrine tumours. Materials & methods: Functional effects and alterations in signalling cascades after treatment with regorafenib were evaluated in myeloid cellline and isolated murine bone marrow macrophages by Western blots, FACS analyses, RT-qPCR and zymography. Results: Regorafenib directly targeted macrophages in vitro in both murine primary macrophages and the monocytic cell line J774. The Colony stimulating factor 1 receptor (CSF1R) signaling pathway was identified as one major target of Regorafenib, which was associated with decreased mTOR/Akt-signalling pathway and decreased inflammatory signaling cascades. Treatment with Regorafenib reduced expression of tumourpromoting and proangiogenic cytokines such as Monocyte chemoattractant protein-1 (MCP-1/CCL2), Monocyte chemoattractant protein-3 (MCP-3/CCL7) and activity of metalloproteinase 9 (MMP9). In addition, Regorafenib directly modulated the functional polarisation of macrophages by affecting its M2 polarisation, thereby restraining the tumourpromoting effects of infiltrating macrophages and decreasing the impact of macrophages as driver of invasiveness and metastasis. Conclusion: Antiangiogenic multikinase inhibitors not only affect tumour cell autonomous and endothelial angiogenic pathways, but also significantly affect the functional phenotype of infiltrating macrophages, thereby reducing their tumour-promoting potential.
signaling in favor of oncogenic properties. Importantly, loss of Smad4 favors complex formation of Smad3 with oncogenic partner proteins. We previously identified the oncogenic Nuclear Factor of activated T cells (NFATc1) transcription factor as a prominent target of oncogenic TGFbsignaling. However, the mechanism of TGFb-controlled NFATc1-dependent transcription in Smad4-deficient PDAC remains elusive. Aims: To characterize NFATc1/Smad3 complex formation, regulation and function in Smad4-deficient pancreatic carcinogenesis and PDAC progression. Materials & methods: NFATc1/Smad3 assembled chromatin complexes are characterized in vitro and in vivo in PDAC cell lines with CRISPR/ Cas9-mediated Smad4 knock out and in Smad4-deficient genetically engineered mice. NFATc1/Smad3 regulated gene signatures are analyzed by genome-wide DNA binding and expression studies. Results: Co-immunoprecipitation experiments revealed Smad4dependent NFATc1/Smad3 transcription complex formation. Reexpression of Smad4 resulted in NFATc1/Smad3 complex disruption, induced apoptosis and decreased cell cycle progression. While TGFb-signaling is not crucial for the formation of the transcription complex, it enhances its stabilization and has an impact on its transcriptional activity. Conclusion: Here we identify nuclear NFATc1/Smad3 complex formation as a key component of oncogenic TGFb-signaling in Smad4-deficient PDAC. Targeting this complex might be a beneficial approach in a subgroup of patients with Smad4-deficient pancreatic cancer.
Abstract ID: 1447. Pancreatic ductal adenocarcinoma (PDAC)-derived exosomes enrich the microenvironment in Myeloid Derived Suppressor Cells (MDSCs) in a Smad4-dependent manner through a new calcium related axis Elisa Gnatta 1, Sara Furlanello 1, Ada Aita 1, Paola Fogar 1, Chiara Frasson 2, Dania Bozzato 1, Andrea Padoan 1, Basso Giuseppe 2, Mario Plebani 1, Daniela Basso 1 1 2
Department of Medicine-DIMED, University of Padova, Italy Department of Woman and Child Health, University of Padova, Italy
Introduction: Genetic aberrations (including Smad4 inactivation) and immunoevasion concur in worsening PDAC prognosis. Calcium signalling plays a relevant role in the elaboration of the adaptive immune response against cancer. Aims: To verify whether PDAC affects immune cells in a Smad4dependent manner and whether tumor-derived exosomes play a part by altering intracellular calcium ([Ca2+]i) fluxes. Materials & methods: 1%FCS-Conditioned media (CM) from two pancreatic cancer cell lines BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) were used. Peripheral blood mononuclear cells (PBMCs) from 8 blood donors were cultured (4 days) in unfractioned control medium, BxPC3/CM and BxPC3-Smad4+/CM, and in Exosomesenriched (pellet), or Exosomes-free (supernatant) media (ultracentrifuCD8+, CD4+CD25+ T-cells, Dendritic gation protocol). CD4+, and two MDSCs subsets cells (CD11b+CD14+DR-¼DCs) (CD11b+CD14-DR+¼mMDSCs; CD11b+CD14-DR-¼gMDSCs) were FACS analyzed. [Ca2+]i fluxes were studied with Fluo-4 stain and recorded by a fluorescent microscope in PBMCs treated in the same conditions. Results: Unfractioned BxPC3-Smad4+/CM significantly induced CD4+CD25+ cells (p¼0.003) while dampening CD8+ cells (p¼0.007) whit respect to control. Both unfractioned PDAC CM significantly dampen DCs (p¼0.009) and gMDSCs (p¼0.013), while they induce mMDSCs (p¼0.006) but only BxPC3-derived exosomes mediate the expansion of both MDSCs populations. Unfractioned BxPC3/CM caused a significant increase in the number of PBMCs with [Ca2+]i peaks (p¼0.009) with respect to control and this was confirmed in Exosomes-enriched CM (p¼ 0.007). After FACS sorted myeloid populations, DCs show a static behaviour while mMDSCs present regular [Ca2+]i peaks. Conclusion: PDAC-derived exosomes from Smad4 expressing, but mainly from Smad4 non-expressing cells, through the increase in [Ca2+]i fluxes, create an immunosuppressive myeloid cells background.
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Abstract ID: 1449, Oral-10. Inhibition of Class I HDAC ameliorates acute and chronic pancreatitis by reducing leukocyte recruitment, acinar-to-ductal metaplasia and fibrosis Marta Bombardo, Enrica Saponara, Ermanno Malagola, Rong Chen, Rolf Graf, Sabrina Sonda Swiss HPB Center, Visceral & Transplantation Surgery, University Hospital Zurich, Switzerland Introduction: Epigenetic mechanisms regulated by the activity of histone deacetylases (HDACs) contribute to the pathology of many inflammatory diseases. Aims: In this study we analyzed whether HDACs are activated during acute (AP) and chronic pancreatitis (CP) and whether pharmacological inhibition of HDACs has a beneficial effect during these diseases. Materials & methods: AP and CP were induced by cerulein injections in wild type C57BL/6 mice. The expression and activity of HDACs and the effects of class I HDAC inhibition with the selective inhibitor MS-275 were evaluated using biochemical, qRT-PCR and imaging techniques. In vitro effects of MS-275 were evaluated using 3D cultures of primary acinar cells and macrophage cell line RAW 264.7. Results: Induction of pancreatitis resulted in increased expression of class I HDACs, increased nuclear HDAC activity and decreased acetylation of nuclear proteins. Inhibitions of class I HDAC with MS-275 significantly reduced pancreatic inflammation both in AP and CP by limiting the migration of neutrophils and macrophages and directly affect macrophage activation. In addition, MS-275 treatment limited the severity of acinar cell damage and prevented the formation of acinar-to-ductal metaplasia, both in vitro and in vivo. Finally, MS-275 reduced the development of fibrosis by down-regulating TGF-b and EGFR signaling in the pancreas. Conclusion: Our investigation demonstrates that class I HDACs play a crucial role in acute and chronic pancreatitis, promoting the development of inflammatory response, metaplastic lesions and tissue fibrosis. In addition, our results show that inhibition of class I HDACs could be a useful treatment for this severe disease.
Abstract ID: 1450. Akt1 regulates the development of inflammation and tissue regeneration during acute pancreatitis Rong Chen 1, Gianluca Mosca 1, Maren Dietrich 2, Enrica Saponara 1, Kamile Grabliauskaite 1, Richard A. Zuellig 2, Oliver Tschopp 2, Rolf Graf 1, Sabrina Sonda 1 1
University Hospital Zurich, Department of Visceral & Transplantation Surgery, Switzerland 2 University Hospital Zurich, Division of Endocrinology, Diabetes and Clinical Nutrition, Switzerland
Introduction: The serine/threonine kinase Akt/PKB plays a key role in the conserved phosphoinositide 3-kinase signaling pathway. In mammals, activation of Akt is implicated in the regulation of divergent cellular processes, including proliferation, survival and cell size. Recently, Akt has also been implicated in the regulation of inflammation through activation of the NF-kB pathway. Three Akt isoforms (Akt1, Akt2, and Akt3) are found in mammals and, despite being highly related and sharing the same structural organization, they show non-redundant physiological functions. Aims: In this study we investigated whether the Akt1 isoform plays a critical role during the development of acute pancreatitis (AP). Materials & methods: AP was induced by repetitive injections of cerulein in wild type (WT) and Akt1-/- mice. The onset and progression of the disease were evaluated in the two mouse strains by serum enzyme quantification, immunohistochemistry and qRT-PCR analyses. Results: Following AP induction, WT and Akt1-/- mice showed comparable initial damage of acinar cells. However, during disease progression, Akt1-/- mice displayed reduced infiltration of inflammatory cells in the
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Abstracts / Pancreatology 16 (2016) S1eS130
pancreas. In addition, lack of Akt1 resulted in reduced acinar cell proliferation and de-differentiation into acinar-to-ductal metaplasia. Molecular analyses revealed a down-regulation of TGF-b signaling in Akt1-/- mice. Conclusion: Our results revealed that Akt1 signaling does not mediate the initial acinar cell damage observed at the onset of AP. However, Akt1 signaling promotes both the inflammatory response and the regeneration of the pancreatic tissue. These findings provide novel insights into the molecular pathways governing pancreatic inflammation and tissue regeneration, with potential therapeutic implications.
Abstract ID: 1451. 1-Deoxy-sphingolipids, novel biomarkers of diabetes, are cytotoxic for exocrine pancreatic cells Rong Chen 1, Thorsten Hornemann 2, Wei Yu 2, Simone Camargo 3, Rolf Graf 1, Sabrina Sonda 1 1 University Hospital Zurich, Department of Visceral & Transplantation Surgery, Switzerland 2 University Hospital Zurich, Institute for Clinical Chemistry, Switzerland 3 University of Zurich, Institute of Physiology, Switzerland
Introduction: Exocrine pancreatic insufficiency and dysfunctions are frequently associated with diabetes mellitus (DM), which also constitutes a risk factor to develop pancreatitis. We recently discovered that deoxysphingolipids (1-deoxySLs) increase during DM and are cytotoxic for beta cells. Aims: Knowing the anatomical and functional relationships between endocrine and exocrine pancreatic tissues, we hypothesize that elevated 1deoxySLs levels observed in DM directly damage the exocrine compartment, thus increasing its predisposition to develop (exocrine) pancreatic diseases. Materials & methods: DM was induced with streptozotocin injections in C57BL/6 mice and Wistar rats. Reduction of 1-deoxySL synthesis was obtained by oral L-serine supplementation. Disease severity was evaluated with biochemical and immunohistochemical methods. Molecular mechanisms of 1-deoxySL-dependent toxicity were evaluated in vitro in pancreatic acinar cells and primary pancreatic fibroblasts. Results: DM induction resulted in increased 1-deoxySL levels but also atrophy and fibrosis of pancreatic parenchima. Reduction of 1-deoxySL synthesis by oral L-serine supplementation ameliorated the damage of the exocrine pancreatic tissue, without restoring insulin production in beta cells. This suggests that elevated 1-deoxySLs rather than insulin deficiency contribute to the exocrine damage in DM. In vitro studies showed that treatment with 1-deoxysphinganine at low micromolar concentration reduced replication and promoted cytotoxicity in pancreatic acinar cells and fibroblasts. Moreover, 1-deoxySL-mediated cytotoxicity was associated with ER stress and ROS production. Conclusion: Our work revealed that 1-deoxySLs are cytotoxic for exocrine pancreatic cells, suggesting a role for these lipids in the exocrine dysfunctions observed following DM. Oral L-serine supplementation could be an option for ameliorating exocrine pancreatic diseases in diabetic patients.
Abstract ID: 1452, Oral-15. The analgesic effect of the mitochondria targeted antioxidant SkQ in experimental pancreatitis Jan D'Haese 1, Maximilian Weniger 1, Leonard Reinelt 1, Jens Neumann 2, Lesca Holdt 3, Matthias Ilmer 1, Bernhard Renz 1, Werner Hartwig 1, Jens Werner 1, Alexandr Bazhin 1 1 LMU Munich, Department of General, Visceral, and Transplantation Surgery, Germany
2 3
LMU Munich, Institute of Pathology, Germany LMU Munich, Institute of Laboratory Medicine, Germany
Introduction: Chronic pancreatitis is one of the main risk factors for pancreatic cancer. In acute and chronic pancreatitis, oxidative stress is thought to play a key role. In this respect, the recently described mitochondria-targeted antioxidant SkQ effectively scavenges reactive oxygen species at nanomolar concentrations. Aims: Therefore, we aimed to characterize the influence of SkQ on tissue injury and pain in acute and chronic pancreatitis. Materials & methods: Both acute and chronic pancreatitis were induced in C57BL/6 mice by intraperitoneal cerulein injections and treatment with SkQ was carried out by peroral applications. Hyperalgesia was assessed by behavioral observation and measurement of abdominal mechanical sensitivity. Blood serum and pancreatic tissue were harvested for analysis of lipase and histology. Results: SkQ did not influence pain, serological or histological parameters of tissue injury in acute pancreatitis. In chronic pancreatitis, a highly significant reduction of pain-related behavior (p < 0.0001) was evident, but histological grading revealed increased tissue injury in SkQtreated animals (p ¼ 0.03). Conclusion: After SkQ-treatment, tissue injury is not ameliorated in acute pancreatitis and increased in chronic pancreatitis. However, we postulate a modest analgesic effect in chronic pancreatitis. Further studies will need to further elucidate the risks and benefits of mitochondria-targeted antioxidants as an analgesic.
Abstract ID: 1453. Non-tumour cell autonomous effects of antiangiogenic multikinase inhibitor regorafenib on infiltrating macrophages in pancreatic neuroendocrine tumours Maren Egidi, Heidi Griesmann, Patrick Michl, Sebastian Krug Department of Gastroenterology and Hepatology, University Hospital, Martin-Luther-University, Halle Saale, Germany Introduction: Pancreatic neuroendocrine tumours (PNETs) represent a distinct hypervascular tumour entity with marked response rates to treatment strategies with antiangiogenic multikinase inhibitors. However, acquired resistance frequently develops and limits the long-term benefit of antiangiogenic approaches. Infiltration of immune cells including tumourassociated macrophages (TAMs) has been associated with tumour progression and increased angiogenesis. Aims: Evaluation of the functional impact of antiangiogenic multikinase inhibitors such as regorafenib on infiltrating macrophages in pancreatic neuroendocrine tumours. Materials & methods: Functional effects and alterations in signalling cascades after treatment with regorafenib were evaluated in myeloid cellline and isolated murine bone marrow macrophages by Western blots, FACS analyses, RT-qPCR and zymography. Results: Regorafenib directly targeted macrophages in vitro in both murine primary macrophages and the monocytic cell line J774. The Colony stimulating factor 1 receptor (CSF1R) signaling pathway was identified as one major target of Regorafenib, which was associated with decreased mTOR/Akt-signalling pathway and decreased inflammatory signaling cascades. Treatment with Regorafenib reduced expression of tumourpromoting and proangiogenic cytokines such as Monocyte chemoattractant protein-1 (MCP-1/CCL2), Monocyte chemoattractant protein-3 (MCP-3/CCL7) and activity of metalloproteinase 9 (MMP9). In addition, Regorafenib directly modulated the functional polarisation of macrophages by affecting its M2 polarisation, thereby restraining the tumourpromoting effects of infiltrating macrophages and decreasing the impact of macrophages as driver of invasiveness and metastasis. Conclusion: Antiangiogenic multikinase inhibitors not only affect tumour cell autonomous and endothelial angiogenic pathways, but also significantly affect the functional phenotype of infiltrating macrophages, thereby reducing their tumour-promoting potential.