- Email: [email protected]
mesenteric inflammation in cirrhotic rats with ascites
POSTER PRESENTATIONS THU-064 Simvastatin neither reduces gut bacterial translocation nor sistemic/mesenteric inflammation in cirrhotic rats with ascites M. Ubeda1,2, L. Muñoz1,2, L. Paule1,2, R. del Campo3, M. Álvarez-Mon1,2,4, A. Albillos1,2,5, A. Albillos. 1Medicine and Medical Specialties, University of Alcalá, Alcalá de Henares; 2CIBERehd, Instituto de Salud Carlos III; 3Microbiology, University Hospital Ramón y Cajal, IRYCIS, Madrid; 4Immune System Diseases and Oncology Service, University Hospital Príncipe de Asturias, Alcalá de Henares; 5Dept of Gastroenterology, University Hospital Ramón y Cajal, IRYCIS, Madrid, Spain E-mail: [email protected] Background and Aims: Clinical studies have shown that statins reduce the risk of decompensation and death in cirrhosis. Statins, such as simvastatin (SIM), correct LPS-driven intrahepatic endothelial dysfunction and inflammation in rats with cirrhosis, and colonic inflammation in mice with colitis. Our hypothesis postulates that SIM exerts its beneficial effect in cirrhosis by reducing gut bacterial translocation (GBT) and the associated intestinal and systemic inflammation. Aim: To investigate the effects of SIM in GBT and intestinal and systemic inflammation in rats with cirrhosis and ascites. Methods: Rats with CCl4-cirrhosis and ascites (n = 14) or controls (n = 6) received a 2-week course of SIM (5 mg/kg.day) or vehicle. The following were measured: GBT and intestinal bacterial overgrowth (IBO) by mesenteric lymph node or feces culture, TNFalpha secreted by peritoneal macrophages by ELISA, systemic and mesenteric inflammation by flow cytometry. Results: Compared with vehicle, SIM did not modify GBT (50% vs. 57%), IBO (8.1 ± 1.3 vs. 7.8 ± 0.7 log10 cfu/g) nor TNFalpha secreted by LPS-stimulated peritoneal macrophages (SIM: from 574 ± 433 to 5762 ± 3602 pg/ml, vehicle: from 426 ± 368 pg/ml to 5144 ± 4583 pg/ ml) in rats with cirrhosis. SIM neither modified systemic and mesenteric inflammation in rats with cirrhosis Peripheral blood % Recently activated Th cells Effector Tc cells Inflammatory monocytes
SIM 6.5 ± 2.2 24.4 ± 9.1 21.1 ± 10.3
Vehicle 7.4 ± 4.6 23.8 ± 8.6 21.6 ± 9.5
Mesenteric lymph nodes SIM
Vehicle
10.5 ± 6.1
7.4 ± 4.1
35.5 ± 8.5 38.02 ± 16.84
33.2 ± 9.2 38.01 ± 14.37
Conclusions: SIM does not reduce GBT neither modulates systemic and mesenteric inflammation in rats with cirrhosis and ascites. The mechanism underlying the beneficial effects of statins in human cirrhosis does not seem to be related with the reduction of GBT or inflammation. THU-065 Osteoprotegerin production and profibrotic activity in liver fibrosis are TGFβ dependent A. Adhyatmika1, K.S.S. Putri2,3, L. Beljaars1, E. Gore2, K.A. Mangnus1, C. Reker-Smit1, E. Post1, K. Poelstra1, P. Olinga2, B.N. Melgert1. 1 Pharmacokinetics, Toxicology, and Targeting; 2Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen; 3 Pharmacy, University of Indonesia, Depok, The Netherlands E-mail: [email protected] Background and Aims: Our previous studies have shown that osteoprotegerin (OPG) is a profibrotic mediator, produced by myofibroblasts, under influence of TGFβ. Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which
cytokines associated with fibrosis induce OPG and through which pathways it can induce fibrotic responses. Methods: Precision-cut liver slices of wild type and STAT6(−/−) mice and 3T3 fibroblasts were used to investigate the effects of TGFβ, IL13, IL1β, and PDGF-BB on expression of OPG and several markers of fibrosis: procollagen 1α1 (Col1α1), α-smooth muscle actin (α-SMA), heat shock protein 47 (HSP47), plasminogen activator inhibitor 1 (PAI1), and fibronectin 2 (Fn2). Results: In addition to TGFβ, only IL13 and not PDGF-BB or IL1β could induce OPG expression in 3T3 fibroblasts and liver slices. This induction was not shown in liver slices of STAT6(−/−) mice and when wild type slices were cotreated with TGFβ receptor 1 kinase inhibitor Galunisertib, suggesting that the OPG-inducing effect of IL13 is mediated through STAT6-dependent TGFβ production, which in turn can induce OPG. In more detail, this IL13-induced TGFβ production is mediated through IL13 receptor α1-activation and subsequent STAT6-dependent upregulation of IL13 receptor α2. The IL13 was then found to signal through the α2 receptor to induce expression of TGFβ. Furthermore, treatment of liver slices with OPG resulted in higher expression of fibrosis markers especially Col-1α1 and TGFβ, which could be inhibited by co-treatment with Galunisertib, indicating the importance of TGFβ in this pathway. Conclusions: IL13 indirectly induces OPG production through STAT6dependent regulation of IL13 receptor α2, which then leads to increased expression of TGFβ followed by upregulation of OPG production. The profibrotic activity of OPG is also dependent on TGFβ. Both results show that OPG and TGFβ are involved in a feed-forward loop during development of liver fibrosis. THU-066 Osteoprotegerin is more than a serum marker in liver fibrosis A. Adhyatmika1, L. Beljaars1, K.S.S. Putri2,3, C. Reker-Smit1, C.E. Boorsma1, B. Guney1, A. Haak1, E. Post1, K. Poelstra1, B.N. Melgert1. 1 Pharmacokinetics, Toxicology, and Targeting; 2Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen; 3 Pharmacy, University of Indonesia, Depok, The Netherlands E-mail: [email protected] Background and Aims: Serum osteoprotegerin (OPG) has clinically been associated with liver fibrosis and has been shown to increase diagnostic accuracy. However, the source and role of OPG is unknown and it is also not clear whether OPG expression responds to spontaneous or drug-induced resolution of fibrosis. Therefore, we aimed to evaluate the expression and biological activities of OPG in fibrotic human and mouse livers and to study its expression in relation to treatment-induced and spontaneous resolution. Methods: Healthy and cirrhotic human liver tissue, liver tissue of healthy mice and mice with CCl4-induced liver fibrosis with or without treatment, murine precision-cut liver slices and 3T3 fibroblasts were analyzed by immunohistochemical staining, ELISA, and real time qPCR. Results: Hepatic OPG levels were significantly higher in human cirrhotic livers and CCl4-induced fibrotic mouse livers compared to healthy livers. Immunohistochemistry confirmed higher hepatic OPG expression in fibrotic tissue and showed localization of OPG in the cirrhotic collagenous bands co-localizing with myofibroblasts (α-smooth muscle actin-positive cells). Experiments with mouse precision-cut liver slices confirmed that liver tissue produces OPG, in particular after stimulation with transforming growth factor-β (TGFβ). Stimulation of 3T3 fibroblasts with TGFβ confirmed fibroblasts as one of the main sources of OPG. Moreover, OPG itself stimulated expressions of collagen-1α1, heat shock protein 47, plasminogen activator inhibitor 1, fibronectin type 2, and importantly transforming growth factor-β, but not α-smooth muscle actin in these liver slices. In vivo, cessation of CCl4 administration or treatment with interferon gamma (IFNγ) in mice led to resolution of fibrosis, which was associated with significantly lower OPG expression in these
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SIM 6.5 ± 2.2 24.4 ± 9.1 21.1 ± 10.3
Vehicle 7.4 ± 4.6 23.8 ± 8.6 21.6 ± 9.5
Mesenteric lymph nodes SIM
Vehicle
10.5 ± 6.1
7.4 ± 4.1
35.5 ± 8.5 38.02 ± 16.84
33.2 ± 9.2 38.01 ± 14.37
Conclusions: SIM does not reduce GBT neither modulates systemic and mesenteric inflammation in rats with cirrhosis and ascites. The mechanism underlying the beneficial effects of statins in human cirrhosis does not seem to be related with the reduction of GBT or inflammation. THU-065 Osteoprotegerin production and profibrotic activity in liver fibrosis are TGFβ dependent A. Adhyatmika1, K.S.S. Putri2,3, L. Beljaars1, E. Gore2, K.A. Mangnus1, C. Reker-Smit1, E. Post1, K. Poelstra1, P. Olinga2, B.N. Melgert1. 1 Pharmacokinetics, Toxicology, and Targeting; 2Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen; 3 Pharmacy, University of Indonesia, Depok, The Netherlands E-mail: [email protected] Background and Aims: Our previous studies have shown that osteoprotegerin (OPG) is a profibrotic mediator, produced by myofibroblasts, under influence of TGFβ. Its expression in experimental models of liver fibrosis correlates well with disease severity and treatment responses. The regulation of OPG in liver tissue is largely unknown and we therefore set out to elucidate which
cytokines associated with fibrosis induce OPG and through which pathways it can induce fibrotic responses. Methods: Precision-cut liver slices of wild type and STAT6(−/−) mice and 3T3 fibroblasts were used to investigate the effects of TGFβ, IL13, IL1β, and PDGF-BB on expression of OPG and several markers of fibrosis: procollagen 1α1 (Col1α1), α-smooth muscle actin (α-SMA), heat shock protein 47 (HSP47), plasminogen activator inhibitor 1 (PAI1), and fibronectin 2 (Fn2). Results: In addition to TGFβ, only IL13 and not PDGF-BB or IL1β could induce OPG expression in 3T3 fibroblasts and liver slices. This induction was not shown in liver slices of STAT6(−/−) mice and when wild type slices were cotreated with TGFβ receptor 1 kinase inhibitor Galunisertib, suggesting that the OPG-inducing effect of IL13 is mediated through STAT6-dependent TGFβ production, which in turn can induce OPG. In more detail, this IL13-induced TGFβ production is mediated through IL13 receptor α1-activation and subsequent STAT6-dependent upregulation of IL13 receptor α2. The IL13 was then found to signal through the α2 receptor to induce expression of TGFβ. Furthermore, treatment of liver slices with OPG resulted in higher expression of fibrosis markers especially Col-1α1 and TGFβ, which could be inhibited by co-treatment with Galunisertib, indicating the importance of TGFβ in this pathway. Conclusions: IL13 indirectly induces OPG production through STAT6dependent regulation of IL13 receptor α2, which then leads to increased expression of TGFβ followed by upregulation of OPG production. The profibrotic activity of OPG is also dependent on TGFβ. Both results show that OPG and TGFβ are involved in a feed-forward loop during development of liver fibrosis. THU-066 Osteoprotegerin is more than a serum marker in liver fibrosis A. Adhyatmika1, L. Beljaars1, K.S.S. Putri2,3, C. Reker-Smit1, C.E. Boorsma1, B. Guney1, A. Haak1, E. Post1, K. Poelstra1, B.N. Melgert1. 1 Pharmacokinetics, Toxicology, and Targeting; 2Pharmaceutical Technology and Biopharmacy, University of Groningen, Groningen; 3 Pharmacy, University of Indonesia, Depok, The Netherlands E-mail: [email protected] Background and Aims: Serum osteoprotegerin (OPG) has clinically been associated with liver fibrosis and has been shown to increase diagnostic accuracy. However, the source and role of OPG is unknown and it is also not clear whether OPG expression responds to spontaneous or drug-induced resolution of fibrosis. Therefore, we aimed to evaluate the expression and biological activities of OPG in fibrotic human and mouse livers and to study its expression in relation to treatment-induced and spontaneous resolution. Methods: Healthy and cirrhotic human liver tissue, liver tissue of healthy mice and mice with CCl4-induced liver fibrosis with or without treatment, murine precision-cut liver slices and 3T3 fibroblasts were analyzed by immunohistochemical staining, ELISA, and real time qPCR. Results: Hepatic OPG levels were significantly higher in human cirrhotic livers and CCl4-induced fibrotic mouse livers compared to healthy livers. Immunohistochemistry confirmed higher hepatic OPG expression in fibrotic tissue and showed localization of OPG in the cirrhotic collagenous bands co-localizing with myofibroblasts (α-smooth muscle actin-positive cells). Experiments with mouse precision-cut liver slices confirmed that liver tissue produces OPG, in particular after stimulation with transforming growth factor-β (TGFβ). Stimulation of 3T3 fibroblasts with TGFβ confirmed fibroblasts as one of the main sources of OPG. Moreover, OPG itself stimulated expressions of collagen-1α1, heat shock protein 47, plasminogen activator inhibitor 1, fibronectin type 2, and importantly transforming growth factor-β, but not α-smooth muscle actin in these liver slices. In vivo, cessation of CCl4 administration or treatment with interferon gamma (IFNγ) in mice led to resolution of fibrosis, which was associated with significantly lower OPG expression in these
Journal of Hepatology 2017 vol. 66 | S95–S332
S147