- Email: [email protected]
P1298 : Hypoxic preconditioning potentiates trophic effects of mesenchymal stem cells on co-cultured human primary hepatocytes
POSTERS P1298 HYPOXIC PRECONDITIONING POTENTIATES TROPHIC EFFECTS OF MESENCHYMAL STEM CELLS ON CO-CULTURED HUMAN PRIMARY HEPATOCYTES H. Qin1,2 , C. Filippi1 , R.R. Mitry1 , A. Dhawan1 , R.D. Hughes1 . 1 Institute of Liver Studies, King’s College Hospital NHS Foundation Trust, 2 Division of Transplantation Immunology & Mucosal Biology, King’s College London School of Medicine, London, United Kingdom E-mail: [email protected] Background and Aims: Hepatocyte transplantation has been emerging as a promising alternative treatment modality for acute liver failure; however, marginal quality of cellular transplants is a major limitation. Mesenchymal stem cells (MSCs) could improve liver-specific metabolism of co-cultured hepatocytes. The present work aimed to potentiate hepatotrophic effects of MSCs co-culture using hypoxic preconditioning (HPc). Methods: Human adipose-derived MSCs were subjected to 20%> (normoxia-preconditioned, NPc) or 2%> O2 (HPc) for 24 h and cocultured with primary human hepatocytes in a 3-dimensional architecture (Figure 1, the arrowheads indicating hepatocytes), with mono-cultured hepatocytes as control. Albumin secretion and CCK18 release were assayed to investigate trophic and antiapoptotic effects of co-culture on hepatocytes. Indirect coculture was established to investigate the role of paracrine factors in hepatotrophic effects of co-culture. Reactive oxygen species (ROS) activity was antagonised by adding N-acetylcysteine to investigate whether HPc potentiated MSCs by intracellular ROS-dependent mechanisms, including TNF-a, TGF-b1, extracellular collagen, and apoptosis-associated caspase and BAX/BCL-2 signalling pathways. Results: HPc significantly potentiated albumin secretion (day 7, HPc vs. NPc, 3.9±0.2 vs. 3.3±0.3 folds of control, P < 0.01) but further inhibited CCK18 release (day 5, 0.44±0.06 vs. 0.63±0.08 fold of control, P < 0.01) from co-cultured hepatocytes. Indirect co-culture showed no significant hepatotrophic effects. HPcinduced potentiative effects were eliminated by ROS antagonisation (albumin, 0.83±0.06 fold of HPc co-culture, P < 0.01; CCK18, 1.4±0.2 folds of HPc co-culture, P < 0.01). Decreased hepatocyte autocrine TNF-a, increased MSCs autocrine TGF-b1 and enhanced MSCs deposition of extracellular collagen mediated potentiative effect of HPc on MSCs co-culture. Enhanced inhibitive effect of HPc on apoptosis of hepatocytes co-cultured with MSCs resulted from downregulated expression of CASP9, BAX, BID and BLK and upregulated expression of BCL-2. Conclusions: HPc potentiated MSCs and improved co-culture hepatotrophic and antiapoptotic effects by nonparacrine, intracellular ROS-dependent mechanisms, including autocrine TGF-b, extracellular collagen and caspases and BAX/BCL-2 signalling pathways.
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P1299 PLATELET ALPHA-GRANULE RELEASE IN LIVER REGENERATION AFTER HEPATECTOMY P. Starlinger1 , S. Haegele1 , D. Wanek1 , S. Zikeli1 , F. Offensperger1 , D. Schauer1 , E. Fleischmann2 , B. Gruenberger3 , C. Brostjan1 , T. Gruenberger1 , A. Assinger4 . 1 Surgery, 2 Anesthesiology, Medical University of Vienna, 3 Internal Medicine, Brothers of Charity Hospital, 4 Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Platelet activation results in a-granule exocytosis and release of mediators crucial for various (patho) physiological processes like angiogenesis and liver regeneration. As a-granules contain pro- and anti-proliferative factors, it has been speculated, that platelets are capable of selective a-granule release. Methods: We investigated if this selective release also occurs in vivo and correlates with clinical outcome by monitoring 130 patients undergoing partial hepatectomy. Results: Liver resection resulted in elevated plasma levels of a-granule factors in all patients. Low plasma levels on the first postoperative day of vascular endothelial growth factor (VEGF) and fibrinogen but high thrombospondin-1 (TSP-1), platelet factor-4 (PF4) and transforming growth factor-b (TGF-b) levels predicted liver dysfunction. Furthermore, platelets of patients with liver dysfunction failed to secrete intra platelet VEGF after liver resection. Based on these results, patients within a high risk group (high TSP-1 and low VEGF) suffered from a significantly increased incidence of postoperative liver dysfunction as well as severe morbidity. Conclusions: This indicates that the postoperative profile of circulating platelet-derived factors correlates with the ability of the remnant liver to regenerate. Accordingly, the modulation of platelet a-granule release in patients may represent an attractive target for therapeutic interventions to modify liver regeneration or angiogenesis. P1300 SPHEROID RESERVOIR BIOARTIFICIAL LIVER TREATMENT INHIBITS ALPHA-AMANITIN INDUCED FULMINANT HEPATIC FAILURE IN RHESUS MONKEY MODEL J. Bao1,2 , H. Bu1,2 , S.L. Nyberg3 . 1 Laboratory of Pathology, 2 Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China; 3 Division of Transplantation Surgery, Department of Surgery, Mayo Clinic, Rochester, United States E-mail: [email protected] Background and Aims: High mortality rate in amanita phalloides intoxications is principally a result of the fulminant hepatic failure (FHF) following massive death of liver cells due to hepatocellular uptake of a-amanitin (a-AMA), the major amatoxin, with limited therapeutic options. Recovery would be more frequent if a supportive therapy were available to correct the toxic milieu of FHF to prevent its extrahepatic manifestations and to assist in liver regeneration. Therefore, a novel supportive therapy, the Spheroid Reservoir Bioartificial Liver (SRBAL) composed of over 100 gram porcine primary hepatocyte aggregates (“spheroids”), was developed. The spheroids were engineered by a novel rocked high-density suspension culture technique. Once formed, spheroids are placed in a continuous perfusion bioreactor, which provides functionality to the device. Methods: The SRBAL was recently evaluated in an ambulatory primate model of a-AMA and LPS combine-induced FHF. Rhesus monkeys were randomized into three treatment groups: no therapy (n = 3), no cell device therapy (n = 3), and SRBAL therapy (n = 3). SRBAL treatment was 6 hours in duration after toxin administration
Journal of Hepatology 2015 vol. 62 | S263–S864
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P1299 PLATELET ALPHA-GRANULE RELEASE IN LIVER REGENERATION AFTER HEPATECTOMY P. Starlinger1 , S. Haegele1 , D. Wanek1 , S. Zikeli1 , F. Offensperger1 , D. Schauer1 , E. Fleischmann2 , B. Gruenberger3 , C. Brostjan1 , T. Gruenberger1 , A. Assinger4 . 1 Surgery, 2 Anesthesiology, Medical University of Vienna, 3 Internal Medicine, Brothers of Charity Hospital, 4 Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria E-mail: [email protected] Background and Aims: Platelet activation results in a-granule exocytosis and release of mediators crucial for various (patho) physiological processes like angiogenesis and liver regeneration. As a-granules contain pro- and anti-proliferative factors, it has been speculated, that platelets are capable of selective a-granule release. Methods: We investigated if this selective release also occurs in vivo and correlates with clinical outcome by monitoring 130 patients undergoing partial hepatectomy. Results: Liver resection resulted in elevated plasma levels of a-granule factors in all patients. Low plasma levels on the first postoperative day of vascular endothelial growth factor (VEGF) and fibrinogen but high thrombospondin-1 (TSP-1), platelet factor-4 (PF4) and transforming growth factor-b (TGF-b) levels predicted liver dysfunction. Furthermore, platelets of patients with liver dysfunction failed to secrete intra platelet VEGF after liver resection. Based on these results, patients within a high risk group (high TSP-1 and low VEGF) suffered from a significantly increased incidence of postoperative liver dysfunction as well as severe morbidity. Conclusions: This indicates that the postoperative profile of circulating platelet-derived factors correlates with the ability of the remnant liver to regenerate. Accordingly, the modulation of platelet a-granule release in patients may represent an attractive target for therapeutic interventions to modify liver regeneration or angiogenesis. P1300 SPHEROID RESERVOIR BIOARTIFICIAL LIVER TREATMENT INHIBITS ALPHA-AMANITIN INDUCED FULMINANT HEPATIC FAILURE IN RHESUS MONKEY MODEL J. Bao1,2 , H. Bu1,2 , S.L. Nyberg3 . 1 Laboratory of Pathology, 2 Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu, China; 3 Division of Transplantation Surgery, Department of Surgery, Mayo Clinic, Rochester, United States E-mail: [email protected] Background and Aims: High mortality rate in amanita phalloides intoxications is principally a result of the fulminant hepatic failure (FHF) following massive death of liver cells due to hepatocellular uptake of a-amanitin (a-AMA), the major amatoxin, with limited therapeutic options. Recovery would be more frequent if a supportive therapy were available to correct the toxic milieu of FHF to prevent its extrahepatic manifestations and to assist in liver regeneration. Therefore, a novel supportive therapy, the Spheroid Reservoir Bioartificial Liver (SRBAL) composed of over 100 gram porcine primary hepatocyte aggregates (“spheroids”), was developed. The spheroids were engineered by a novel rocked high-density suspension culture technique. Once formed, spheroids are placed in a continuous perfusion bioreactor, which provides functionality to the device. Methods: The SRBAL was recently evaluated in an ambulatory primate model of a-AMA and LPS combine-induced FHF. Rhesus monkeys were randomized into three treatment groups: no therapy (n = 3), no cell device therapy (n = 3), and SRBAL therapy (n = 3). SRBAL treatment was 6 hours in duration after toxin administration
Journal of Hepatology 2015 vol. 62 | S263–S864