Yelena Cheporko

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure.

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1β levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaβ (NF-ĸ β) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1β, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IĸB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-ĸB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.

Bax ablation protects against hepatic ischemia/reperfusion injury in transgenic mice

Apoptosis appears to be a central mechanism of cell death following reperfusion of the ischemic liver. The aim of this study was to determine the effect of decreased expression of the proapoptotic Bax gene on hepatic apoptotic warm ischemia/reperfusion (I/R) injury. Three groups of mice were studied: homozygotic knockout mice (Bax−/−); heterozygotic (Bax+/−); and wild type (Bax+/+). Isolated mouse livers were subjected to 90 minutes of ischemia (37°C) followed by 15 minutes of reperfusion. Bax and Bcl‐2 expression in liver tissue homogenates was measured by Western blot. Serum liver enzyme levels were measured and intrahepatic caspase‐3 activity was determined by fluorimetric assay. Oil red O (ORO) staining was performed for fat detection. Apoptotic cells were identified by morphological criteria, immunohistochemistry for caspase‐3, and terminal deoxynucleotidyl transferase‐mediated 2′‐deoxyuridine 5′‐triphosphate nick‐end labeling (TUNEL) assay. At 1 minute of reperfusion, the ischemic (Bax−/−) livers were characterized by statistically significantly lower liver enzyme levels and lower caspase‐3 activity than the ischemic (Bax+/+) livers (P < 0.05 for both). The reduction in postischemic apoptotic hepatic injury in the ischemic Bax−/− livers group was confirmed morphologically, by the significantly reduced microvesicular steatosis as determined by ORO staining, fewer apoptotic hepatocyte cells detected (P < 0.05); immunohistochemically, by the significantly weaker activation of caspase‐3 compared to the ischemic group (P < 0.05); and by TUNEL assay (P < 0.05). Similar levels of antiapoptotic Bcl‐2 protein expression were detected in all 3 groups of ischemic livers on Western blots. Bax protein was not expressed in Bax‐deficient livers and was detected in Bax+/+ normal livers. In the Bax+/− livers, levels of the damage markers were moderate. In conclusion, The better tolerance of Bax knockout livers to I/R injury suggests that the Bax gene may serve as a potential target for therapeutic intervention in hepatic I/R injury. Liver Transpl 13:1181–1188, 2007.

Effect of adenosine A2A receptor agonist (CGS) on ischemia/reperfusion injury in isolated rat liver.

Ischemia/reperfusion injury during liver transplantation is a major cause of primary nonfunctioning graft for which there is no effective treatment other than retransplantation. Adenosine prevents ischemia-reperfusion-induced hepatic injury via its A2A receptors. The aim of this study was to investigate the role of A2A receptor agonist on apoptotic ischemia/reperfusion-induced hepatic injury in rats. Isolated rat livers within University of Wisconsin solution were randomly divided into four groups: (1) continuous perfusion of Krebs-Henseleit solution through the portal vein for 165 minutes (control); (2) 30-minute perfusion followed by 120 minutes of ischemia and 15 minutes of reperfusion; (3) like group 2, but with the administration of CGS 21680, an A2A receptor agonist, 30 μ g/100 ml, for 1 minute before ischemia; (4) like group 3, but with administration of SCH 58261, an A2A receptor antagonist. Serum liver enzyme levels were measured by biochemical analysis, and intrahepatic caspase-3 activity was measured by fluorometric assay; apoptotic cells were identified by morphological criteria, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) fluorometric assay, and immunohistochemistry for caspase-3. Results showed that at 1 minute of reperfusion, there was a statistically significant reduction in liver enzyme levels in the animals pretreated with CGS (p < 0.05). On fluorometric assay, caspase-3 activity was significantly decreased in group 3 compared to group 2 (p < 0.0002). The reduction in postischemic apoptotic hepatic injury in the CGS-treated group was confirmed morphologically, by the significantly fewer apoptotic hepatocyte cells detected (p < 0.05); immunohistochemically, by the significantly weaker activation of caspase-3 compared to the ischemic group (p < 0.05); and by the TUNEL assay (p < 0.05). In conclusion, the administration of A2A receptor agonist before induction of ischemia can attenuate postischemic apoptotic hepatic injury and thereby minimize liver injury. Apoptotic hepatic injury seems to be mediated through caspase-3 activity.

Erythropoietin increases survival and attenuates fulminant hepatic failure injury induced by D-galactosamine/lipopolysaccharide in mice.

Background. Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), d-galactosamine (GalN)-induced FHF is a well-established model of liver injury in mice. Erythropoietin has a powerful tissue-protective effect in animal models. The aim of this study was to investigate the effect and mechanism of recombinant human erythropoietin (rhEPO) administration in FHF mice. Methods. C57BL/6 (n=42) mice were studied in vivo in a fulminant model induced by GalN/LPS. rhEPO was administered 30 min after the induction of FHF. Serum liver enzymes and hepatic tumor necrosis factor (TNF)-&agr; and interleukin (IL)-1&bgr; levels were determined. Histologic analysis was performed, and apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor (NF)-&kgr;B and c-Jun-N-terminal kinase (JNK) activation were studied using Western blot analysis. Results. After the induction of FHF, all control mice died within 12 hr of GalN/LPS administration. However, 83% of mice that were administered rhEPO were alive 2 weeks later, and overall survival improved (Kaplan-Meier, P<0.001). The serum liver enzymes, hepatic TNF-&agr; and IL-1&bgr; levels, liver histologic injury, and apoptotic hepatocytes were significantly reduced in FHF mice that were administered rhEPO compared with untreated mice. A significant decrease in hepatic NF-&kgr;B and JNK activation was noted in FHF rhEPO-treated mice compared with FHF untreated mice. Conclusions. The administration of rhEPO brought about increased survival and attenuation of the hepatic injury. This was associated with decreased hepatic NF-&kgr;B and JNK activation and thus TNF-&agr; and IL-1&bgr; levels. These findings have important implications for the potential use of rhEPO in FHF.

Dual effect of erythropoietin on liver protection and regeneration after subtotal hepatectomy in rats

The only currently offered curative option for many patients with primary or secondary liver tumors is the resection of hepatic tumors. The aim of this study was to evaluate the role of recombinant human erythropoietin (rhEPO) in liver protection and regeneration after subtotal hepatectomy in rats. Rats undergoing 70% hepatectomy received an intraperitoneal injection of saline (control) or rhEPO (4 U/g) 30 minutes prior to resection. Liver function was assessed by the measurement of the international normalized ratio (INR) levels, and hepatic injury was assessed by serum alanine aminotransferase and aspartate aminotransferase levels. Hepatic apoptosis was assessed by intrahepatic caspase‐3 activity and morphological criteria. The regeneration capacity of remnant livers was assessed over 7 days with the regenerated liver/body weight ratio, immunohistochemistry markers of cell proliferation (Ki‐67) and angiogenesis (von Willebrand factor), and phosphorylated extracellular signal‐regulated kinase signaling. Two and 4 days after subtotal hepatectomy, the regenerated liver/body weight ratio was significantly higher in animals treated with rhEPO versus the control group (P < 0.005). Serum liver enzymes and INR levels on days 2 and 4 post‐hepatectomy were significantly lower in animals pretreated with rhEPO in comparison with the control group (P < 0.005). No statistically significant difference was noted in intrahepatic hepatic caspase‐3 activity, immunohistochemistry for caspase‐3, or a terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling assay between the hepatectomized groups. In the rhEPO‐pretreated group, the mitotic index, Ki‐67 and von Willebrand factor expression, and extracellular signal‐regulated kinase activity were significantly higher on day 2 post‐hepatectomy (P < 0.05) in comparison with the control group. In conclusion, rhEPO treatment may offer a unique beneficial dual‐function strategy for hepatic protection and regeneration immediately after subtotal hepatectomy in rats. Liver Transpl, 2010.

Ras inhibition attenuates myocardial ischemia-reperfusion injury

Myocardial injury, developed after a period of ischemia/reperfusion (I/R) results in the destruction of functional heart tissue, this being replaced by scar tissue. Intracellular signaling pathways mediating cardiomyocyte death are partially understood and involve the activation of Ras. p38-MAPK, JNK and Mst-1 are downstream effectors of Ras protein. We hypothesized that S-farnesylthiosalicylic acid (FTS), a synthetic small molecule that detaches Ras from the inner cell membrane, consequently inhibiting Ras activity, reduces I/R myocardial injury in vitro and in vivo. Wistar rat hearts were isolated, mounted on the Langendorff apparatus and subjected to ischemia (30 min, 37 degrees C) and reperfusion. During the reperfusion period, the hearts were perfused with FTS (1 microM) solution or control buffer. Left anterior descending (LAD) ligation and subsequent reperfusion was performed in two groups of Wistar rats. Rats received 5mg/kg FTS or PBS according to two protocols: (A) FTS or PBS were administered daily 7 days prior, immediately before and 14 days (every other day) after LAD occlusion or (B) every other day for 14 days post-I/R. Hearts from FTS-treated rats (Langendorff) and FTS-treated rats (protocol A) showed a significant improvement in myocardial performance and smaller scar tissue compared with the PBS group. Infarct size in the FTS-treated group was 12.7+/-2% vs. 23.7+/-4% in the PBS-treated (in vitro) group and 17.3+/-2.5% vs. 36+/-7% compared with control I/R rats (in vivo) p<0.05. These effects may be associated with the down regulation of JNK as a short-term effector and with Mst-1 in the long-term remodeling process.

The role of excessive versus acute administration of erythropoietin in attenuating hepatic ischemia-reperfusion injury.

Ischemia-reperfusion injury (I/R) is the main cause of primary graft nonfunction. Our aim was to evaluate the effect of excessive versus acute administration of erythropoietin (EPO) in attenuating the hepatic injury induced by I/R in mice. The effect of segmental (70%) hepatic ischemia was evaluated in a transgenic mouse line with constitutive overexpression of human EPO cDNA and in wild-type (WT) mice. Mice were randomly allocated to 5 main experimental groups: (i) WT-sham, (ii) WT ischemia, (iii) WT ischemia + recombinant human erythropoietin (rhEPO), (iv) transgenic-sham, and (v) transgenic ischemia. The EPO-pretreated mice showed a significant reduction in liver enzyme levels and intrahepatic caspase-3 activity and fewer apoptotic hepatocytes (p < 0.05 for all) compared with the WT untreated I/R group. EPO decreased c-Jun N-terminal kinase (JNK) phosphorylation and nuclear factor-κB (NF-κB) expression during I/R. In transgenic I/R livers, baseline histology showed diffused hepatic injury, and no significant beneficial effect was noted between the WT untreated and the transgenic I/R mice. In conclusion, acute pretreatment with EPO in WT mice attenuated in vivo I/R liver injury. However, in excessive EPO overexpression, the initial liver injury abolished the beneficial effect of EPO. These findings have important implications for the potential use of acute EPO in I/R injury during liver transplantation.

Uridine-5'-triphosphate protects against hepatic- ischemic/reperfusion injury in mice.

Background and Aim. Mitochondrial calcium overload triggers apoptosis and also regulates ATP production. ATP and uridine-5′-triphosphate (UTP) depletion from hepatic tissue after ischemia causes cell death. ATP and UTP binds to cell membranes of the hepatocytes through P2Y receptors. Our aim was to investigate the role of UTP on the hepatic injury induced by ischemia. Methods. Isolated mouse livers were randomly divided into five groups: (1) control group; (2) ischemic group (90 min); (3) as group 2, but with the administration of UTP; (4) as group 2, but with the administration of suramin, a P2Y antagonist; and (5) as group 3, but with the simultaneous administration of suramin and UTP. Results. There was a postischemic significant reduction in the release of liver enzymes in the animals pretreated with UTP, the intrahepatic caspase-3 activity was significantly decreased, and the intrahepatic ATP content increased compared with group 2 (ischemic untreated). UTP prevented intracellular Ca2+ overload after hypoxia in hepatocyte cultures. In the UTP-treated groups, significantly fewer apoptotic hepatocyte cells were noted by weaker activation of caspase-3 and by the transferase-mediated dUTP nick end labeling assay. The administration of suramin prevented the beneficial effect of endogenous ATP. UTP treatment attenuated the degradation of I&kgr;Bα (nuclear factor-kappaB inhibitor) by 80% during reperfusion with no effect on c-Jun N terminal kinase phosphorylation. Conclusion. The administration of UTP before induction of ischemia-reperfusion can attenuate hepatic injury. UTP administration decreased cytosolic Ca2+ overload in hypoxic conditions. UTP-mediated protective effects may be regulated through nuclear factor- kappaB inactivation. These findings have important implications for the potential use of UTP in ischemic hepatic injury.

236 BONE MARROW AND NON-BONE MARROW TLR4 REGULATES ACUTE HEPATIC INJURY INDUCED BY ENDOTOXEMIA

236 BONE MARROW AND NON-BONE MARROW TLR4 REGULATES ACUTE HEPATIC INJURY INDUCED BY ENDOTOXEMIA Z. Ben Ari, O. Avlas, O. Pappo, Y. Cheporko, L. Bachmatov, R. Zemel, A. Shainberg, E. Hochhauser. Liver Disease Center, Sheba Medical Center Tel Hashomer, Ramat Gan, Liver Research Laboratory, Felsenstein Medical Research Center, Tel Aviv University, Petah Tiqwa, Gonda (Goldschmied) Medical Diagnostic Research Center, The Mina & Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Department of Histopathology, Sheba Medical Center Tel Hashomer, Ramat Gan, Israel E-mail: gbenari@bezeqint.net