Dominique Crenesse

A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis

Apoptosis is activated during the early phase of reperfusion after liver ischemia and after liver transplantation in animals. However, the molecular basis of ischemia‐induced cell death remains poorly understood. In this study we show that hepatocytes from ischemic liver lobes undergo apoptosis after reperfusion. In vivo pretreatment of rats with a specific inhibitor of caspases abrogates the apoptotic response in ischemic liver lobes. Inhibition of apoptosis can be accounted for by total inhibition of caspase activation as assessed in an enzymatic assay and by specific affinity labeling. Treatment with a caspase inhibitor fully protects rats from death induced by ischemia/ reperfusion. These findings indicate that liver injury after ischemia/reperfusion can be prevented by inhibition of caspases. Thus, caspase inhibitors may have important therapeutic implications in liver ischemic diseases and after liver transplantation.—Cursio, R., Gugenheim, J., RicciJ, E., Crenesse, D., Rostagno, P., Maulon, L., Saint‐Paul, M.‐C., Ferrua, B., Auberger, P. A caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis. FASEB J. 13, 253–261 (1999)

Matrix metalloproteinase inhibition protects rat livers from prolonged cold ischemia–warm reperfusion injury

Matrix metalloproteinases (MMPs) have been implicated in the hepatic injury induced after cold ischemia–warm reperfusion (CI‐WR), by altering the extracellular matrix (ECM), but their precise role remains unknown. The hepatic MMP expression was evaluated after 2 conditions of CI (4°C for 24 and 42 hours: viable and nonviable livers) followed by different periods of WR, using isolated perfused rat livers. CI‐WR induced moderate changes in hepatic MMP transcript levels not influenced by CI duration, whereas gelatinase activities accumulated in liver effluents. Therefore, the protective effect of a new phosphinic MMP inhibitor, RXP409, was tested after prolonged CI. RXP409 (10 μM) was added to the University of Wisconsin solution, and livers were preserved for 42 hours (4°C), then reperfused for 1 hour in Krebs solution (37°C), containing 20% erythrocytes. Liver viability parameters were recorded, and the extent of cell necrosis was evaluated on liver biopsies, using trypan blue nuclear uptake. Treatment with RXP409 significantly improved liver function (transaminase release and bile secretion) and liver injury. In particular, the MMP inhibitor significantly modified the extent of cell death from large clusters of necrotic hepatocytes as found in control livers (2%–60% of liver biopsies; mean, 26% ± 9%) to isolated necrotic hepatocytes as found in treated livers (0.2%–12%; mean, 3% ± 2%) (P < 0.05). Conclusion: These data demonstrate that MMPs, by altering the ECM, play a major role in liver CI‐WR injury leading to extensive hepatocyte necrosis and that their inhibition might prove to be a new strategy in improving preservation solutions. (HEPATOLOGY 2007.)

Caspase inhibition protects from liver injury following ischemia and reperfusion in rats

Abstract Normothermic ischemia and reperfusion of the liver results in microcirculatory failure followed by necrosis and cell death. Recently, another type of cell death, apoptosis or programmed cell death, was found to be activated during the early phase of reperfusion after liver ischemia. Caspases are cysteine proteinases specifically involved in the initiation and execution phases of apoptosis. The aim of this study was to demonstrate that inhibition of apoptosis by a specific inhibitor of caspases might protect the liver against ischemia/reperfusion injury. Rats were divided into three groups: group 1, control, PBS administration; group 2, Z‐Asp‐cmk (Z‐Asp‐2,6‐dichlorobenzoyl‐oxymethylketone) treatment; group 3, sham‐operated control animals. Z‐Asp‐cmk (0.5 mg Z‐Asp‐cmk dissolved in 300 μl PBS solution containing 1 % DMSO) was injected intravenously, 2 min prior to induction of 120 min ischemia. Survival rates were compared and serum activities of aspartate aminotransferases and alanine aminotransferases were assessed in the blood collected from the suprahepatic vena cava. Histology of the liver was assessed 6 h after the end of ischemia. Apoptosis was detected by the terminal deoxynucleotidyl transferase‐mediated dUTP‐FITC nick end‐labeling method (TUNEL method) and by electrophoresis for analysis of DNA fragmentation. Caspase activity was determined by measuring hydrolysis of the CPP32‐ like substrate Ac‐DEVD‐pNA and absorption of paranitroaniline. Z‐Asp‐cmk treatment significantly increased 7‐day survival (95 %) compared with that in nontreated rats (30%, P < 0.001). Serum activities of aminotransferases and the extent of liver congestion and necrosis were significantly (P < 0.001) decreased after treatment with Z‐Asp‐cmk. TUNEL‐positive cells were detected 3‐6 h after reperfusion in the control group. In Z‐Asp‐cmk pretreated rats, a dramatic decrease in the number of TUNEL‐positive cells was observed. Analysis of DNA fragmentation of freshly isolated hepatocytes confirmed these results. Caspase activity was increased 3‐6 h after reperfusion in the control group, but significantly (P < 0.001) decreased after treatment with Z‐Asp‐cmk. These findings demonstrate that liver injury following ischemia and reperfusion can be prevented by inhibition of caspases. Caspase inhibitors may have important implications for therapy in liver disease and after liver transplantation.

Rat liver ischemia-reperfusion-induced apoptosis and necrosis are decreased by FK506 pretreatment

The aim of this study was to demonstrate that tacrolimus (FK506) has a hepatoprotective effect by reducing ischemia-reperfusion-induced apoptosis and necrosis, both of which lead to post-surgical liver dysfunction. An ischemia-reperfusion model and primary cultured rat hepatocytes subjected to hypoxic and reoxygenation phases, mimicking the surgical process, were used. c-Jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK1/SAPK1) activation leads to caspase 3 activation, a trigger of apoptosis. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation or Western-blotting experiments. Apoptosis was assessed by measuring caspase activation and by TUNEL (terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate-biotin nick-end labeling) reaction. Necrosis was assessed histologically. Tacrolimus improved the survival rate of rats subjected to ischemia-reperfusion. After FK506 pretreatment, the liver necrosis rate was reduced, and ischemia-reperfusion-induced JNK1/SAPK1 activation and apoptosis were significantly decreased. In hypoxia-reoxygenation-subjected hepatocytes, tacrolimus reduced JNK1/SAPK1 and caspase 3 activation. In the liver, tacrolimus prevented ischemia-reperfusion-induced apoptosis and necrosis.

Distinct epithelial gene expression phenotypes in childhood respiratory allergy.

Epithelial cell contribution to the natural history of childhood allergic respiratory disease remains poorly understood. Our aims were to identify epithelial pathways that are dysregulated in different phenotypes of respiratory allergy. We established gene expression signatures of nasal brushings from children with dust mite-allergic rhinitis, associated or not associated with controlled or uncontrolled asthma. Supervised learning and unsupervised clustering were used to predict the different subgroups of patients and define altered signalling pathways. These profiles were compared with those of primary cultures of human nasal epithelial cells stimulated with either interleukin (IL)-4, IL-13, interferon (IFN)-&agr;, IFN-&bgr; or IFN-&ggr;, or during in vitro differentiation. A supervised method discriminated children with allergic rhinitis from healthy controls (prediction accuracy 91%), based on 61 transcripts, including 21 T-helper cell (Th) type 2-responsive genes. This method was then applied to predict children with controlled or uncontrolled asthma (prediction accuracy 75%), based on 41 transcripts: nine of them, which were down-regulated in uncontrolled asthma, are directly linked to IFN. This group also included GSDML, which is genetically associated with asthma. Our data revealed a Th2-driven epithelial phenotype common to all children with dust mite allergic rhinitis. It highlights the influence of epithelially expressed molecules on the control of asthma, in association with atopy and impaired viral response.

Warm ischemia‐reperfusion injury is decreased by tacrolimus in steatotic rat liver

Ischemia‐reperfusion (I‐R) injury is poorly tolerated by fatty livers, most probably secondary to reduced cellular adenosine triphosphate (ATP) levels. We investigated the effectiveness of tacrolimus pretreatment on fatty liver I‐R injury in obese Zucker rats. Tacrolimus (0.3 mg/kg, intravenously) was injected 24 hours before a 75‐minute ischemic period and rats were sacrificed 6 hours later. Tacrolimus modified the response to I‐R observed in obese Zucker rats, when compared to nontreated obese rats: a significant reduction in hepatocyte necrosis was associated with a significant increase in hepatocyte apoptosis. In addition, cell necrosis and apoptosis were significantly and inversely correlated in lean nontreated and treated obese Zucker rats following I‐R. Tacrolimus also significantly increased the hepatic ATP levels, reduced in nontreated obese rats, toward values found in lean Zucker rat livers. This protective effect of tacrolimus was further confirmed in vivo by a significantly improved survival following pretreatment with tacrolimus, 24 hours prior to ischemia. In conclusion, in obese Zucker rat livers, tacrolimus pretreatment reversed the I‐R injury toward the one found in lean Zucker rats. The correlations between ATP levels and the opposite changes in necrosis and apoptotic pathways strongly suggest a cause‐effect relationship between tacrolimus and changes in ATP levels. Liver Transpl 12:217–225, 2006.

Diltiazem Reduces Apoptosis in Rat Hepatocytes Subjected to Warm Hypoxia-Reoxygenation

Interruption of hepatic blood flow is necessary in surgery, but the liver is sensitive to ischemia and reperfusion. Hypoxia induces an increase in intracellular calcium concentration. In previous studies, we have shown that hypoxia-reoxygenation (H/R) increased calcium influx and induced JNK1/SAPK1 activation which was involved in the triggering of apoptosis. The aim of this study was to demonstrate that diltiazem, a calcium inhibitor, reduced JNK1/SAPK1 activation and consequently could decrease H/R-induced apoptosis. Experiments were performed, in the presence of diltiazem, on primary cultured rat hepatocytes, subjected to warm H/R phases and in a liver ischemia-reperfusion model. The activation status of JNK1/SAPK1 was evaluated by immunoprecipitation and immunohistolocalisation experiments, while apoptosis was assessed by measuring caspase activity and by TUNEL labeling. Diltiazem inhibited H/R-induced JNK1/SAPK1 activation and decreased apoptosis. It could be used to improve postoperative liver function.

Hypoxia‐reoxygenation‐induced chemokine transcription is not prevented by preconditioning or intermittent hypoxia, in mice hepatocytes

Prolonged ischemia used in liver surgery and/or transplantation causes cellular damage resulting in apoptosis and necrosis. Ischemia–reperfusion (I/R) led Kupffer cells to pro‐inflammatory cytokines secretion [tumor necrosis factor (TNF)‐α, interleukin‐1] which involve chemokines secretion by hepatocytes. These chemokines have neutrophil chemotactic properties and neutrophils are involved in the development of I/R‐induced necrosis. The aim of this study was to specify the consequence of partial oxygen pressure variation on hepatocyte chemokines synthesis and to verify if intermittent hypoxia and/or preconditioning could decrease it. It was performed on primary cultured mice hepatocytes and Kupffer cells, subjected to continuous, intermittent hypoxia or preconditioning phases, mimicking surgical processes. The chemokine secretion was evaluated by RNase protection assay and enzyme‐linked immunosorbent assay method. Only monocyte chemoattractant protein‐1 (MCP‐1) and macrophage inflammatory protein‐2 (MIP‐2) mRNA formation were observed, especially after 1‐h hypoxia followed by 10‐h (for MCP‐1) or 24‐h reoxygenation (for MIP‐2). In conclusion, TNF‐α and coculture with Kupffer cells increased hepatocyte chemokines mRNA transcription, whereas surgical split up protocols (intermittent hypoxia and preconditioning) had no significant effect.

Inhibition of calcium influx during hypoxia/reoxygenation in primary cultured rat hepatocytes.

Calcium has been demonstrated to play an important role in hepatocyte damage during ischemia/reperfusion phases. Calcium influx was determined in primary cultured rat hepatocytes submitted to a succession of warm hypoxia and reoxygenation phases in the presence of diltiazem, gallopamil and a Na+/H+ antiport inhibitor, HOE-694. Only diltiazem significantly inhibited calcium influx with higher potency after reoxygenation than after hypoxia only, suggesting a complex mechanism of action of diltiazem which could act on different physiological functions involved in Ca2+ invasion of hepatocytes after hypoxic insult.

Evaluation of impulse oscillometry during bronchial challenge testing in children.

The impulse oscillation system (IOS) allows easy measurement of respiratory system impedance (Zrs). The aim of this retrospective study was to evaluate the accuracy of IOS parameters obtained during methacholine challenge by comparison with “the gold standard” forced expiratory volume in the first second (FEV1).