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(The FASEB Journal. 1999;13:253-261.)
© 1999 FASEB

RESEARCH COMMUNICATION

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

RAFFAELE CURSIOa , JEAN GUGENHEIMa , JEAN EHRLAND RICCIe , DOMINIQUE CRENESSEa , PHILIPPE ROSTAGNOc , LAURENCE MAULONe , MARIE-CHRISTINE SAINT-PAULd , BERNARD FERRUAb and PATRICK AUBERGER 1 ,e


a Laboratoire de Recherches Chirurgicales,

b Laboratoire de Parasitologie, Université de Nice-Sophia-Antipolis, Faculté de Médecine, 06107 Nice Cedex 2, France;

c Laboratoire de Cytométrie, Centre Antoine Lacassagne, 06189 Nice-Cedex 2, France;

d Service d'Anatomo-Pathologie, Hôpital Pasteur, Faculté de Médecine, 06000 Nice, France; and

e CJF INSERM 96.05 `Activation des cellules hématopoiétiques`, Faculté de Médecine, 06107 Nice, France


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptosisis 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., Ricci, J. 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.


Key Words: caspases • tumor necrosis factor • aminotransferases • DNA fragmentation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HEPATIC ISCHEMIA OCCURS in a variety of circumstances, including liver transplantation (1) , hemodynamic or cardiogenic shock (2) , and during liver resection for trauma or tumor (3) . This ischemia/reperfusion injury results in microcirculatory failure, followed by necrosis and cell death (4) . Recently, another type of cell death, apoptosis or programmed cell death (5) , has been found to occur in various types of tissue and organ damage caused by ischemia reperfusion and transplantation 6-8) . In fact, apoptosis was found to be activated during the early phase of reperfusion after liver ischemia in rat (9) and after liver transplantation in pig (10) . Furthermore, apoptosis is involved in endothelial cell damage during conservation and is influenced by organ storage solution (11) . Apoptosis and necrosis may occur in parallel, both contributing to cell death in liver disease. However, the extent of apoptotic cell death is frequently underestimated since apoptosis is a rapid event with an estimated duration of 2–3 h; only scattered single cells may be affected and apoptotic bodies, which are readily eliminated, are small (12) . Caspases are cysteine proteinases specifically involved in the initiation and execution phases of apoptosis (13) . This has been demonstrated by studies showing that inhibitors of this class of proteinases block essentially all forms of apoptosis in vitro 13-20) and that caspase overexpression induces apoptosis in various cell lines (13 , 21 , 22 ). Tumor necrosis factor (TNF)2 receptor and Fas ligation are two well-documented processes leading to massive apoptosis in various cellular models 23-28) . As inhibition of caspases results in protection against TNF-{alpha}- and Fas-mediated apoptosis in vitro, activation of apoptosis during the reperfusion phase after normothermic liver ischemia was evaluated morphologically and biochemically in rats pretreated or not with the caspase inhibitor Z-Asp-2,6-dichlorobenzoyl-oxymethylketone (Z-Asp-cmk).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal preparation and hepatic ischemia procedure
All experiments were conducted in accordance with institutional guidelines for the care and use of laboratory animals. Male Lewis rats (LEW RTI1) weighing 250—300 g were purchased from the CNRS-CNSEAL (Orléans La Source, France). For each experiment there was less than 25 g difference between animals. The rats, housed individually in Plexiglas cages, were allowed free access to food and water before, during, and after the ischemia. The animal rooms were windowless with temperature (22±2°C) and lighting controls (light on at 07.00 h and off at 21.00 h; 14 h light/10 h dark). All experiments started between 8 and 11 AM. A segmental normothermic ischemia of the liver was induced as described by Baker (29) . Briefly, the anterior abdominal wall was shaved and prepped with povidone-iodine (Betadine) solution. The abdomen was entered through a midline incision under ether anesthesia and ischemia induced by occluding the blood vessels, including the bile duct to the median and left lateral lobes, with an atraumatic vascular clamp. After 120 min of warm ischemia, the vascular clamp was released. This procedure was considered to render ischemia in 70% of liver tissue by weight (30) . The abdomen was closed in two layers with silk. Sham-operated animals underwent manipulation of the liver and mobilization of the relevant vessels, but had no clamp application. After the operation animals were kept in individual cages. At adequate interval times after the end of surgical procedure, the animals were killed by exsanguination or anesthetized before hepatocyte isolation. Necropsy was performed on all animals to control absence of surgically related complications.

Inhibitors and substrates
Stock IL-1ß converting-enzyme inhibitor III (Z-Asp-cmk) (31 , 32 ) was dissolved in 100% DMSO to a final concentration of 50 mg/ml. Z-Asp-cmk, acetyl-DEVD-pNA and biotinyl-DEVD-CHO were purchased from (Alexis Biochemicals).

Experimental groups
Rats, prepared as described above, were divided into two groups of 20 rats each. Group 1 animals (control group) were injected intravenously, via dorsal penile vein, with 300 µl of a phosphate-buffered saline (PBS) solution containing 1% DMSO 2 min prior to induction of the ischemia. Animals in group 2 were injected intravenously, via dorsal penile vein, with 0.5 mg of Z-Asp-cmk, dissolved in 300 µl of a PBS solution containing 1% DMSO, 2 min prior to induction of ischemia. In these conditions it is assumed that most of the inhibitor will be retained in the ischemic lobes.

Mortality study
Mortality rates were assessed at day 7. Ten sham-operated animals were included as controls.

Measurement of aminotransferases
Another set of animals was prepared as described above for measurement of aminotransferases. Blood samples for measurement of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were collected via the in-dwelling venous line 3 to 6 h after the end of ischemia from control or Z-Asp-cmk-treated rats (10 rats for each group) and quantitated using standard clinical automated analysis. Blood sampling was also performed in three sham-operated control animals.

Histological studies
Specimens were fixed in 10% formalin and embedded in paraffin. Sections at 3 µm intervals were stained with hematoxylin and eosin. Six hours after reperfusion, the extent of sinusoidal congestion and liver necrosis was semiquantitatively assessed in 45 samples of median and left lateral lobes as follows: congestion: none = 0, minimal = 1, mild = 2, moderate = 3, severe = 4; liver necrosis: none =0, single-cell necrosis = 1, up to 30% lobular necrosis = 2, up to 60% lobular necrosis = 3, more than 60% lobular necrosis = 4. Blind analysis was carried out for all histological studies. To detect apoptotic cells, liver tissue was taken at 0, 1, 3, 6, and 12 h after the end of ischemia. Three rats per group were used at each interval of time studied. Livers were excised and tissues were immediately cryopreserved. Six micrometer sections were prepared for the terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling method (TUNEL method) of Gavrieli et al. (see ref 29 ) with minor modifications. Briefly, the sections were fixed with 4% paraformaldehyde in PBS for 30 min according to standard protocols. After 2 rinses in PBS buffer, tissue sections were incubated on ice for 2 min in permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate). After rinses in PBS endogenous peroxidase, activity was blocked by 0.3% H2O2 in methanol for 30 min at room temperature. After two rinses in PBS buffer, 50 µl TUNEL reaction mixture (Boehringer-Mannheim, Mannheim, Germany) was added to samples, which were incubated in a humidified chamber for 1 h at 37°C. Sections were rinsed in PBS twice. After addition of 50 µl of antifluorescein antibody conjugated with peroxidase for 30 min, treated sections were treated with DAB (Boehringer-Mannheim) for 5 min at room temperature and stained with hematoxylin Harris. Each section was examined by light microscopy at 40 high-power fields. The morphology of hepatocytes in situ was also examined on paraffin-embedded tissue section using both TUNEL and propidium iodide labeling. Two to three hundred hepatocytes on sections were examined and TUNEL-positive hepatocytes were counted. The number of TUNEL-positive hepatocytes per 100 hepatocytes was calculated. To avoid potential error in statistical sampling, fields were randomly selected. Histological examination was performed by one of the authors in a blind manner. A negative control was included in each experiment by performing the same procedure without terminal transferase.

Isolation of hepatocytes and flow cytometric analysis
To detect apoptotic cells, livers were digested with collagenase at 0, 1, 3, 6, and 12 h after the end of ischemia. At each interval studied, three rats per group were used. Briefly, hepatocytes were isolated from rats with collagenase (Sigma type IV, Sigma Chemical, St. Louis, Mo.) by the perfusion method (33) , modified as described (34) . Dissociated hepatocytes were then collected in William's culture medium. Cell viability, assessed by trypan blue exclusion, was averaged.

DNA fragmentation
Freshly isolated hepatocytes from nonischemic and ischemic liver lobes prepared from untreated or Z-Asp-cmk-treated rats were lysed with 400 µl of lysis buffer (10 mM Tris, pH 7.5, 5 mM EDTA, and 0.2% Triton-X-100). Lysates were treated for 30 min with 100 µg/ml RNAse and then incubated for 30 min with 100 µg/ml proteinase K, as described previously (35) . Cellular DNA was ethanol-precipitated, dried, and resuspended in Tris-EDTA buffer (10 mM Tris pH 7.5, 5 mM EDTA). DNA was separated by electrophoresis on 1.2% agarose, then stained with ethidium bromide. In some experiments, freshly isolated hepatocytes were further cultured in vitro for 1 to 24 h in the presence or the absence of TNF-{alpha} or TNF-{alpha} plus cycloheximide; DNA fragmentation was assessed as described above.

Caspase assay and affinity labeling of caspases
After isolation of hepatocytes, cytosols were prepared at 4°C and immediately assayed for enzymatic activity. Briefly, cells were sedimented at 1000 x g for 5 min, washed in PBS, resuspended in buffer A (25 mM tris/HCl pH 7.5, 5 mM MgCl2, 1 mM EGTA, 1 mM PMSF, 10 µg/ml pepstatin, 10 µg/ml leupeptin), and sonicated. Cell extracts were sedimented at 20,000 x g for 30 min. After addition of dithiothreitol to a final concentration of 2 mM, caspase activity was assayed immediately. Briefly, 100 µg of cytosolic proteins in 50 µl buffer A were diluted with 150 µl buffer A supplemented with 0.15% Triton-X-100 and incubated at 37°C with 200 µM of either acetyl-DEVD-pNA or acetyl-YVAD-pNA in 96-well microtiter plates. At different times, hydrolysis activities were determined by the measure of absorbance of para-nitroaniline at 405 nm. For affinity labeling, aliquots containing 50 µl of hepatocyte cytosolic proteins (2 mg/ml) in a final volume of 100 µl were incubated 1 h at 37°C with 0.25 mM biotinyl-DEVD-CHO. Extracts were then diluted three times in 3x concentrated sodium dodecyl sulfate (SDS) sample buffer, heated to 95°C for 5 min, subjected to electrophoresis on 12.5% polyacrylamide gels, transferred to PVDF membranes, probed with peroxidase-labeled streptavidin (1/10,000), and visualized by enhanced chemiluminescence (Amersham, Arlington Heights, Ill.). In some experiments, caspase 3 (17 and 20 kDa bands) was also visualized by immunoblotting.

Statistical analysis
Significance for mortality results was assessed using the Chi2 test. Results were expressed as mean ±SEM. The comparison for statistical significance was performed according to the Kruskal-Wallis test for serum activities of aminotransferases and histological parameters. Statistical significance was set at P <0.05. Error bars in figures represent standard errors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Z-Asp-cmk protects rats from lethal ischemia/reperfusion
To evaluate the effect of caspase inhibition on ischemia-induced liver apoptosis, rats were injected with 0.5 mg Z-Asp-cmk or vehicle alone (1% DMSO in sterile PBS) 2 min before occlusion of the blood vessels. Immediately after occlusion of the hepatic vessels, the anterior lobes became pale. After releasing the clamp, the liver turned dark and rapidly gained its normal color. The degree of liver necrosis and congestion was significantly lower in the Z-Asp-cmk-treated group than that in the control group (1.8±0.6 vs. 3.6±0.6 and 1.3±0.4 vs. 3.5±0.9, for groups 2 and 1, respectively), P <0.001 (Fig. 1 ). The localization of necrosis was heterogeneous, predominating in subcapsular and mediolobular areas. No evidence of inflammatory reaction was seen. Histological lesions were absent in the sham-operated control animals (not shown). AST and alanine ALT levels increased by 3 to 6 h after the end of the ischemic period (Fig. 2 ), but the release of liver enzyme was markedly lower in animals treated with Z-Asp-cmk (AST: 4.099 ±1.461 UI/L and ALT: 4.522 ±1006 UI/L) compared to the control group (AST: 12.144 ±2.543 UI/L and ALT: 13.032 ±2.607 UI/L, P<0.001) after a 6 h reperfusion period. In sham-operated control animals, the aminotransferases serum levels were 193 ±41 UI/L for AST and 211 ±20 UI/L for ALT. Finally, most of the Z-Asp-cmk-treated rats (95%, 19 of 20, P<0.001) survived and were healthy 6 months after the onset of the experiments, whereas 70% of untreated rats (14 of 20) died within the first 24 h after ischemia (Table 1 ). No deaths were observed in sham-operated group. On necropsy, severe necrosis of the liver was present in all untreated rats. The antiapoptotic effect of Z-Asp-cmk was confirmed by the TUNEL assay (36) , which detects DNA fragmentation in situ. Six hours after reperfusion numerous hepatocytes from ischemic liver lobes were TUNEL positive (Fig. 3 A), whereasvirtually no TUNEL-positive liver cells were observed in the nonischemic liver lobe counterparts (not shown). In Z-Asp-cmk pretreated rats there was a dramatic decrease in the number of TUNEL-positive cells (Fig. 3B ). To confirm the apoptotic mode of death, the morphology of cells was examined on paraffin-embedded tissue sections. As shown in Fig. 3 , morphology of apoptotic hepatocytes was readily distinguishable by the condensed and fragmented state of their nuclei (Fig. 3C ). DNA fragmentation in situ was virtually abolished in ischemic lobes from Z-Asp-cmk-treated rats (Fig. 3D ). Indeed, in Z-Asp-cmk pretreated rats no apoptotic nuclei were observed as judged by propidium iodide labeling and TUNEL assay (Fig. 3D ), whereas hepatocyte nuclei showed extensive chromatin condensation and fragmentation in untreated rats (Fig. 3C ). The yellow coloration of chromatin observed in Fig. 3C is due to the colocalization of FITC (TUNEL) and propidium iodide. The inhibitory effect of the caspase inhibitor was confirmed by quantitative analysis of hepatocyte apoptosis. Indeed (as shown in Fig. 3E ), more than 40% of the cells were TUNEL positive 3 to 6 h after liver reperfusion in untreated rats vs. 2 to 3% in Z-Asp-cmk-treated animals.



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  		Figure 1. Numerical degree of liver necrosis and congestion, 6 h after reperfusion, in control and Z-Asp-cmk-treated groups. Rats were treated or not with Z-Asp-cmk, as described above. Numerical degree of liver necrosis and congestion was determined as described in Materials and Methods. A significant difference was observed between the two groups (P<0.001). Each group consisted of 10 rats. Each point is the mean ±SEM of 45 determinations.



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  		Figure 2. Serum AST and ALT levels after a 120 min normothermic hepatic ischemia 6 h after reperfusion. Animals were treated or not with Z-Asp-cmk 2 min prior to a 120 min normothermic hepatic ischemia period. Six hours after reperfusion, blood samples were collected and aminotransferase levels were evaluated as described in Materials and Methods. Ten rats were used for each determination. Three sham-operated rats were also included in the protocol. Results are the mean ±SEM of 10 determinations made in triplicate. Treatment decreased significantly aminotransferase serum levels compared with control group (P<0.001).


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  		Table 1. Effect of Z-Asp-cmk on the survival rate of ischemic ratsa



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  		Figure 3. Determination of liver apoptosis in situ by the TUNEL method. A, B) Histological analysis of liver sections stained with hematoxylin. Terminal deoxyribonucleotidyl transferase d-UTP nick-end labeling method was carried out on 6 µm section prepared from ischemic liver lobes obtained from control (A) or Z-Asp-cmk-treated rats (B) after a 6 h reperfusion period. Morphology of hepatocytes was also examined on 6 µm paraffin-embedded tissue sections prepared from control (C) or Z-Asp-cmk-treated animals (D), respectively, as described in Materials and Methods. E) Quantitative analysis of apoptotic hepatocytes in hepatic liver lobes from treated or untreated animals 6 h after ischemia/reperfusion. The number of apoptotic cells was determined by cell counting. Controls ({square}); Z-Asp-cmk ().

Biochemical characterization of apoptosis in ischemic livers
Freshly isolated hepatocytes from ischemic or nonischemic liver lobes prepared from control or Z-Asp-cmk-treated rats were analyzed for DNA fragmentation immediately after dissociation of the liver by the collagenase perfusion technique (Fig. 4 ). In untreated rats no evidence of apoptosis was observed in hepatocytes prepared from nonischemic hepatic lobes (lane 1), whereas massive internucleosomal DNA fragmentation was found to occur in cells from ischemic lobes after a 3 h reperfusion period (lane 2). The caspase inhibitor had no effect of its own on nonischemic liver lobes (lane 3), but fully protected cells derived from ischemic liver lobes from apoptosis (lane 4). Identical results were obtained after a 6 h reperfusion period (not shown). Twelve to 24 h after reperfusion, the protective effect of the inhibitor on DNA fragmentation, although significant, was however reduced (not shown).



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  		Figure 4. DNA fragmentation in liver lobes from control and Z-Asp-cmk-treated rats. Rats were injected or not with Z-Asp-cmk 2 min before a 120 min normothermic hepatic ischemia. Three hours after reperfusion, hepatocytes from both groups of rats were isolated by the collagenase perfusion method. DNA fragmentation was evaluated immediately on freshly isolated hepatocytes prepared from nonischemic or ischemic liver lobes.

Implication of caspases in ischemia/reperfusion-induced hepatocyte death
As proteases encoded by the caspase gene family are required for the initiation and execution phases of apoptosis 13-20) , caspase activity was assessed after a 3 and 6 h reperfusion period by measuring hydrolysis of the CPP32-like substrate Ac-DEVD-pNA. Caspase activity increased by 8- to 10-fold in hepatocytes prepared from ischemic liver lobes (Fig. 5 ). Pretreatment of rats by Z-Asp-cmk prior to ischemia/reperfusion abrogated caspase activity (Fig. 5) . No evidence of Ac-YVAD-pNA hydrolysis was obtained in identical conditions (not shown), indicating that the protective effect of Z-Asp-cmk on ischemia/reperfusion-induced liver cell apoptosis is mediated by inhibition of CPP32-like caspase activities. To confirm the role of caspases in this model of ischemia/reperfusion, cellular lysates prepared from isolated hepatocytes derived from control or Z-Asp-cmk-treated rats were incubated with biotinyl-DEVD-CHO. Cellular extracts were then analyzed by monodimensional SDS-polyacrylamide gel electrophoresis, followed by Western blotting with peroxidase-labeled biotin. Affinity labeling with this inhibitor selectively modifies the large subunits of active caspases 37-39) . Activation of at least four caspase bands with subunits of 24, 22, 20, and 17 kDa was clearly detectable in cell extracts derived from isolated hepatocytes prepared from ischemic liver lobes (Fig. 6 A, B, lane 2). No evidence of caspase activation was detected in ischemic liver lobes from Z-Asp-cmk-treated rats (Fig. 6A . B, lane 4). Z-Asp-cmk by itself failed to affect caspase labeling in nonischemic liver lobes (Fig. 6 , lane 3). Identical results were obtained after a 6 h reperfusion period (Fig. 6B ).



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  		Figure 5. Caspase activation during normothermic liver ischemia and reperfusion. After isolation of hepatocytes, cytosols were prepared at 4°C and immediately assayed for enzymatic activity. Caspase activity was measured in the presence of 0. 2 mM acetyl-DEVD-pNA in a final volume of 200 µl in 96-well microtiter plates. Caspase activity was determined in the presence or the absence of an excess of acetyl-DEVD-CHO (10 µM). Caspase activity was determined at different times by measuring the absorbance of para-nitroaniline at 405 nm. The caspase assay was performed after a 3 h (A) and 6 h (B) reperfusion period, respectively. Results are expressed as nanomoles acetyl-DEVD-pNA hydrolyzed/min and per milligram of proteins.



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  		Figure 6. Affinity labeling of caspases. Aliquots containing 50 µl of hepatocyte cytosolic proteins (2 mg/ml) in a final volume of 100 µl were incubated for 1 h at 37°C with 0.25 µM biotinyl-DEVD-CHO. Extracts were then diluted three times in 3x concentrated SDS buffer sample, heated to 95°C for 5 min, subjected to electrophoresis on 12.5% polyacrylamide gels, transferred to PVDF membranes, probed with peroxidase-labeled streptavidin, and visualized by enhanced chemiluminescence (Amersham). A, B) Cell extracts were prepared from rat livers after a 3 and 6 h reperfusion period, respectively. Ischemia: 2 h.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Most studies of apoptosis have been performed in cell culture, but only recently has the role of apoptosis been considered in integrated organ models such as the liver. Although several investigators have recently reported activation of apoptosis during the reperfusion phase after liver and cardiac ischemia 7-9) , the biochemical mechanisms underlying apoptosis in these models have not been studied so far, nor has it been shown whether caspase inhibition can protect rats from death induced by ischemia/reperfusion, as is the case in Fas- and TNF-{alpha}-mediated fulminant liver destruction 40-47) . Using a rat model of ischemia reperfusion in this study, we provided both morphological and biochemical evidence of hepatocyte apoptosis during the early phase of liver reperfusion after a 2 h period of ischemia. Cell death was activated as soon as 1 h after reperfusion, and maximal cell death occurred within 3 to 6 h after reperfusion. Caspase activation was maximal at 3 h and preceded ischemia/reperfusion-induced liver apoptosis as assessed by both an enzymatic assay using Ac-DEVD-pNA as substrate and affinity labeling with biotinyl-DEVD-CHO. In a recent report, activation of apoptosis has been measured by the TUNEL method during the reperfusion phase after rat liver ischemia (after an ischemic period of 30 and 60 min) (29) . In this study, maximal cell death was also observed 3 to 6 h after reperfusion. To the best of our knowledge, we provide here the first biochemical demonstration that CPP32-like caspases are activated after normothermic reperfusion of the liver. We show that administration of the caspase inhibitor Z-Asp-cmk, 2 min prior to ischemia, efficiently protects rats from lethal liver injury that normally occurs 24–48 h after surgery. Indeed, in the presence of the caspase inhibitor, the survival rate of rats was increased from 30% to 95%. The protective effect of Z-Asp-cmk can be totally accounted for by caspase inhibition since this inhibitor was found to abolish ischemia-mediated activation of caspases, as determined in an enzymatic assay and by specific affinity labeling of caspase large subunits. Surprisingly, despite a total inhibition of caspase activities and DNA fragmentation in ischemic liver lobes upon Z-Asp-cmk treatment, the level of transaminases, though drastically reduced, remained relatively high. One possible explanation would be that the caspase inhibitor fully blocked the apoptotic events consecutive to ischemia reperfusion, but acted less efficiently on the liver necrosis that often accompanied ischemia/reperfusion. Nevertheless, our results demonstrate that the sole inhibition of caspases is sufficient to protect rats from lethal injury consecutive to ischemia/reperfusion.

It has recently been shown that intraperitoneal injection of Z-VAD-cmk fully protects mice from Fas-induced liver apoptosis in vivo, whereas Ac-YVAD-cmk was less potent and Z-Asp-cmk was ineffective in inhibiting liver apoptosis under identical conditions (41) . The apparent discrepancy between our results and those described by Rodriguez et al. (41) is surprising since an identical concentration of inhibitor was used in both studies (0.5 mg). However, in our model the caspase inhibitor was injected 2 min prior to ischemia. It was assumed that, under these conditions, most of the inhibitor was distributed and remained in the liver at the time of ischemia. Thus, the local concentration of Z-Asp-cmk in our model of ischemia/reperfusion is likely to be significantly higher as compared to the concentration used in the latter study.

The mechanism by which ischemia/reperfusion leads to liver injury is presently not understood, even if hepatic cell death in this model is probably multifactorial. Release of inflammatory cytokines such as TNF-{alpha}, Fas/Fas ligand interaction, and oxygen-free radicals alone or in combination have been proposed to contribute to the destruction of liver cells after reperfusion. All these signals are thought to converge in the activation of caspases, the executioners of apoptosis. It is now well established that caspase activation is a prerequisite for many forms of cell death and that blockade of caspases in various cell lines and some animal models is generally sufficient to inhibit apoptosis induced by different stimuli. The pivotal role of caspases in the regulation of apoptosis is likely to explain why Z-Asp-cmk is so efficient in protecting rats from lethal normothermic ischemia/reperfusion in the present study.

TNF-{alpha} is a potent mediator of hepatocyte apoptosis both in vitro and in vivo when sensitizing concentrations of actinomycin D or cycloheximide are present. Liver reperfusion after ischemia is associated with elevated circulating levels of TNF-{alpha} (48) . Anti-TNF antiserum has been described to decrease transaminase levels after normothermic ischemia/reperfusion, suggesting that TNF could be implicated in the physiopathologic alterations consecutive to this process (48) . However in the latter study, survival rates were not significantly different in groups of rats treated with or without anti-TNF serum. Thus, hepatic cell death after normothermic reperfusion of the liver is unlikely to be the consequence solely of TNF-{alpha} release, as also suggested previously for the development of hepatitis (42) .

In conclusion, to the best of our knowledge, we show for the first time that in vivo inhibition of caspases can fully protect rats from lethal liver injury induced by ischemia/reperfusion. Many processes such as TNF-{alpha}/TNF receptor and Fas/Fas ligand interaction and oxygen-free radical generation have been implicated in liver cell death. Inhibiting one of these pathways individually is probably not sufficient to prevent liver damage. In contrast, blockade of caspases, which represents the point of convergence of these different pathways, could offer an interesting strategy for the treatment of different liver pathologies. Indeed, ischemia/reperfusion occurs in a variety of circumstances, including liver transplantation. Thus, caspase inhibitors may have important therapeutic applications not only in the treatment of fulminant hepatitis, as suggested previously (40 , 41 ), but also in liver ischemic diseases and as adjuvants for liver preservation in hepatic transplantation and surgery.



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  		Figure .


   ACKNOWLEDGMENTS
 
This work was supported by the Institut National de la Santé et de la Recherche Medicale and a grant from la ligue Nationale contre le Cancer. We are indebted to Dr. Ellen Van-Obberghen Shilling for reviewing the manuscript.


   FOOTNOTES
 
1 Correspondence: CJF INSERM 96.05 Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail auberger{at}unice.fr

2 Abbreviations: AST, aspartate aminotransferase; ALT, alanine aminotransferase; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TNF, tumor necrosis factor; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end-labeling method; Z-Asp-cmk, Z-Asp-2,6-dichlorobenzoyl-oxymethylketone.

Received for publication July 10, 1998. Revision received October 6, 1998.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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