1 First Department of Surgery, 2 Department of Laboratory Medicine, and 3 Department of Molecular Oncology and Angiology, Research Center on Aging and Adaptation, Shinshu University School of Medicine, Nagano 390-8621, Japan
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ABSTRACT |
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Apoptosis plays an important role in liver ischemia and reperfusion (I/R) injury. However, the molecular basis of apoptosis in I/R injury is poorly understood. The aims of this study were to ascertain when and how apoptotic signal transduction occurs in I/R injury. The apoptotic pathway in rats undergoing 90 min of warm ischemia with reperfusion was compared with that of rats undergoing prolonged ischemia alone. During ischemia, mitochondrial cytochrome c was released into the cytosol in a time-dependent manner in hepatocytes and sinusoidal endothelial cells, and caspase-3 and an inhibitor of caspase-activated DNase were cleaved. However, apoptotic manifestation and DNA fragmentation were not observed. After reperfusion, nuclear condensation, cells positive for terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling, and DNA fragmentation were observed and caspase-8 and Bid cleavage occurred. In contrast, prolonged ischemia alone induced necrosis rather than apoptosis. In summary, our results show that release of mitochondrial cytochrome c and caspase activation proceed during ischemia, although apoptosis is manifested after reperfusion.
apoptosis; mitochondria; inhibitor of caspase-activated deoxyribonuclease
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INTRODUCTION |
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ISCHEMIA AND REPERFUSION (I/R) injury in the liver is of clinical importance in humans after hemorrhagic and cardiogenic shock, liver surgery, or liver transplantation. It is increasingly recognized that apoptosis occurs in I/R injury models of the liver (20, 32). In particular, a caspase inhibitor reduced rat liver I/R injury as indicated by a reduction of serum aspartate aminotransferase levels to one-third of levels found in nontreated rats (7). This suggests a pivotal role of apoptotic pathways in liver I/R injury.
Numerous morphological and biochemical studies, including terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) and DNA laddering, showed that hepatocytes and sinusoidal endothelial cells (SECs) undergo apoptosis soon after reperfusion (1-3 h) (20, 32). Until now, it has been suggested that reperfusion may initiate hepatocyte and SEC apoptosis. However, because these morphological and biochemical changes appear at the final phase in apoptosis, apoptosis may be triggered during ischemia. So far, no reports have addressed apoptotic changes during ischemia. Also, no one has described when apoptosis begins and what induces the apoptotic cascade in hepatocytes and SECs during I/R injury in vivo.
At least two main pathways execute apoptosis. Both share activation of effector caspases, specifically, caspase-3 (35, 36). Activated caspase-3 cleaves caspase substrates, such as an inhibitor of caspase-activated DNase (ICAD), during the execution phase of apoptosis (10, 30). ICAD exists as a complex with a caspase-activated DNase (CAD) that promotes apoptotic DNA fragmentation, and cleavage of ICAD releases the active CAD (10, 30).
The first pathway involves the mitochondria (mitochondrial pathway). Cytotoxic reagents, radiation, growth factor deprivation, and hypoxia activate it (1, 3, 4). These stimuli cause the release of cytochrome c from the mitochondria into the cytosol. The released cytochrome c activates caspase-9, in concert with the cytosolic factor dATP (or ATP) and apoptotic protease-activating factor-1, and subsequently activates caspase-3 via proteolytic processing (24, 25, 44). The second pathway is stimulated by cell surface death receptors such as tumor necrosis factor (TNF) receptor 1 and Fas. Ligation of their ligands to the receptor leads to caspase-8 activation, with subsequent activation of caspase-3 (death receptor pathway) (35).
Recently, it has been reported that Bid, a BH3 domain-containing proapoptotic Bcl-2 family member, is cleaved and activated by caspase-8. In turn, the cleaved Bid induces the release of cytochrome c from mitochondria (23, 26).
This study was conducted to determine when and how the apoptotic cascade is initiated and the pathway through which apoptosis proceeds. We examined chronological changes in mitochondrial cytochrome c release, activation of caspase-8 and -3, and cleavage of Bid and ICAD in a well-documented rat model of warm I/R liver injury. General caspase inhibitor was also used to confirm our results.
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MATERIALS AND METHODS |
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Antibodies.
The following primary antibodies were obtained: anti-cytochrome
c monoclonal antibody (clone 7H8.2C12, PharMingen, San
Diego, CA and clone 6H2.B4, Promega, Madison, WI); anti-p11 subunit of caspase-3 (K19), Bid, and ICAD (Santa Cruz Biotechnology, Santa Cruz,
CA); anti-caspase-8 (Chemicon, Temecula, CA); anti-cytochrome oxidase
subunit IV (Molecular Probes, Eugene, OR); anti-TNF- (R&D Systems,
Minneapolis, MN); and anti-actin (Sigma, St. Louis, MO).
Rat model of liver I/R injury. Forty male Wistar rats (Japan SEC, Hamamatsu, Japan) weighing 250-300 g were used. All animals were maintained under standard conditions and fed water and rodent chow ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of Shinshu. The animals were fasted for 12 h before surgery but were allowed access to water. Rats were anesthetized by inhalation of halothane (Takeda Chemical Industries, Osaka, Japan). A complete midline incision was made. All structures (hepatic artery, portal vein, and bile duct) leading to the left and median liver lobes (~70% of liver mass) were occluded with a microvascular clamp for 90 min. This method of partial hepatic ischemia allows for portal decompression through the right and caudate lobes and thus prevents mesenteric venous congestion. Reperfusion was initiated by removal of the clamp. The abdomen was closed in two layers, and the animals were returned to their cages. Rats were killed at 0 (control; n = 5), 15 (n = 5), 30 (n = 5), and 90 (n = 5) min after the start of the ischemia and at 1 (n = 5) and 3 (n = 5) h after reperfusion. To examine the roles of reperfusion, ischemia was prolonged for up to 180 (n = 5) and 270 min (n = 5) without reperfusion. Samples of liver tissue were obtained and preserved for the experiments described below.
Inhibition of caspase activity. To inhibit caspase activity, 0.3 mg of Z-Val-Ala-Asp(OMe)-fluoromethylketone (ZVAD-fmk; Enzyme System Products, Livermore, CA) in 1% DMSO was injected intravenously via the dorsal penile vein 2 min before induction of ischemia. Rats were killed either 90 min after the start of the ischemia or 3 h after reperfusion (n = 5/experimental condition).
Measurement of serum alanine aminotransferase and TNF-.
Blood samples were collected from the abdominal aorta. The serum
samples were centrifuged, and supernatants were stored at
80° until
used. After completion of the experiment, serum alanine aminotransferase (ALT) levels were measured with an automated serum
analyzer (Olympus, Tokyo, Japan). Plasma TNF-
levels were measured
with a commercially available rat TNF-
ELISA kit (R&D Systems)
according to the manufacturer's instructions.
TUNEL staining. To detect cells undergoing apoptosis, the tissue sections were stained via the TUNEL procedure (13), with some modifications. Briefly, the liver tissue was immediately fixed in 8% paraformaldehyde at 4°C for 20-22 h and embedded in paraffin. The tissue was sectioned at 4 µm, dewaxed, rehydrated, and digested with 20 µg/ml of proteinase K (Sigma). Endogenous peroxidase was blocked by treatment in 0.3% hydrogen peroxidase. The sections were then rinsed in water and incubated with 50 µl of terminal deoxynucleotidyl transferase (TdT) buffer (30 mmol/l Tris · HCl, pH 7.2, 140 mmol/l sodium cacodylate, and 1 mmol/l cobalt chloride) containing 8.3 U of TdT (Boehringer Mannheim, Mannheim, Germany) and 0.83 nmol biotinylated 16-dUTP (Boehringer Mannheim) in a moist chamber at 37°C for 60 min. The sections were then rinsed and incubated with horseradish peroxidase-conjugated streptavidin (DAKO, Glostrup, Denmark), diluted 1:500 in 0.01 mol/l Tris · HCl (pH 7.5) plus 150 mmol/l NaCl (TBS) containing 1% BSA (Sigma), for 30 min at room temperature. They were then rinsed in TBS and stained with diaminobenzidine.
DNA fragmentation. DNA fragmentation was assayed by agarose gel electrophoresis (33) with some modifications. Frozen liver samples were minced, and 1 µl of sample was lysed with 20 µl of lysis buffer (50 mmol/l Tris · HCl, pH 7.8, 10 mmol/l EDTA, and 0.5% SDS). Lysates were treated with 10 mg/ml of proteinase K at 60°C for 90 min and then incubated with 10 mg/ml of RNase (Sigma) for 30 min. After brief centrifugation of the lysates, the supernatants were separated by electrophoresis on 1.5% agarose gels with 0.5 µg/ml of ethidium bromide. Hinc II-digested X174 (TOYOBO, Tokyo, Japan) was used as a molecular weight standard. The DNA fragmentation pattern was examined on photographs taken under ultraviolet illumination.
Histological study. Formalin-fixed, paraffin-embedded tissue was sectioned at 4 µm and stained with hematoxylin and eosin, and the morphological aspects of cell death were evaluated.
Isolation of the mitochondrial and cytosolic fractions. For Western blot analysis and ELISA, protein from both the mitochondrial and cytosolic fractions was extracted as follows. Samples were gently homogenized at 800 rpm with a speed-controlled mechanical skill drill (B-100; Tokyo Rikakikai, Tokyo, Japan) by douncing five times in a Teflon-glass Potter-Elvehjem homogenizer (Sanshyo, Tokyo, Japan) in 9 volumes of buffer A (20 mmol/l HEPES-KOH, pH 7.5, 250 mmol/l sucrose, 10 mmol/l KCl, 1.5 mmol/l MgCl2, 1 mmol/l EDTA, 1 mmol/l EGTA, 1 mmol/l dithiothreitol, 0.1 mmol/l phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 5 µg/ml of pepstatin). The homogenates were centrifuged at 800 g at 4°C for 10 min, then at 8,000 g at 4°C for 10 min. The 8,000-g pellets were washed with buffer A, made soluble in buffer B (10 mmol/l Tris · HCl, pH 8.0, 0.5% Nonidet P-40, and 5 mmol/l CaCl2), and were then used as the mitochondrial fraction. The supernatant was further centrifuged at 100,000 g for 60 min at 4°C in an ultracentrifuge (Beckman Coulter, Fullerton, CA). The resulting supernatant was used as the soluble cytosolic fraction.
Western blotting. Liver tissue was homogenized in a buffer containing 20 mmol/l Tris · HCl, pH 7.5, 150 mmol/l NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate, 2 mmol/l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride, 2 µg/ml of aprotinin, 10 µg/ml of leupeptin, and 5 µg/ml of pepstatin. The homogenates were centrifuged at 12,000 g for 10 min at 4°C, and the supernatants were collected. Protein concentration was measured with a bicinchoninic acid protein assay (BCA protein assay kit; Pierce, Rockford, IL). The same amounts of protein from liver homogenates, the cytosolic fraction, and the mitochondrial fraction were dissolved in sample buffer (25 mmol/l Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, 0.02% bromphenol blue, and 3% 2-mercaptoethanol), loaded on 12.5 or 14% polyacrylamide gels, and electrophoresed. Proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA) by electroblotting. Membranes were blocked for 1 h at room temperature with 5% nonfat dried milk and 0.1% BSA in TBS containing 0.1% (vol/vol) Tween 20 (TBS-T) and then were incubated for 1 h with primary antibodies diluted in TBS-T containing 5% fetal bovine serum (FBS). After being washed in TBS-T three times, the membranes were incubated for 1 h with peroxidase-conjugated sheep anti-mouse antibody (Amersham Pharmacia Biotech), sheep anti-rabbit antibody (Amersham), or donkey anti-goat antibody (Chemicon) diluted in TBS-T containing 5% FBS. After another wash in TBS-T, the blots were developed by enhanced chemiluminescence (ECL; Amersham) and exposed to X-ray film (RX-U; Fuji, Kawasaki, Japan).
Quantification of cytochrome c in liver tissue. To quantify the cytochrome c released from mitochondria, the same amount of protein from the cytosolic fraction of the samples mentioned in Western blotting was measured with a commercially available cytochrome c ELISA kit (MBL, Nagoya, Japan) according to the manufacturer's instructions.
Immunohistochemistry.
Liver samples were placed in freezing medium (OCT compound; Sakura,
Tokyo, Japan). This was snap-frozen in an acetone bath cooled in liquid
nitrogen. The specimens were stored at 80°C until they were
sectioned. Frozen sections were cut at 5 µm and placed on glass
slides coated with poly-L-lysine. Sections were air-dried
for 30 min and fixed for 15 min in cold acetone. The fixed sections
were washed in TBS, blocked with 1% BSA, and incubated with an
anti-cytochrome c or anti-cytochrome oxidase subunit IV antibody, at a dilution of 1:50 in 1% BSA overnight at 4°C, and with
biotinylated anti-mouse antibody (DAKO) and fluorescein
isothiocyanate-conjugated streptavidin (DAKO). The sections were then
studied under a fluorescence microscope (BX50; Olympus).
Measurement of caspase activity in liver tissue.
Caspase-3 or -8 activity was measured with commercially available
caspase-3 or -8 fluorometric protease assay kits (MBL) according to the
manufacturer's instructions. Briefly, liver tissue was homogenized in
a lysis buffer. The homogenates were centrifuged at 12,000 g
for 10 min at 4°C, and the supernatants were collected. The same
amounts of protein from the liver homogenates were dissolved in the
lysis buffer. The samples were incubated at 37°C with
N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Ac-DEVD-AFC) or
N-acetyl-Ile-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin (Ac-IEDT-AFC) in the presence or absence of the specific casapse-3 or
-8 inhibitor (Ac-DEVD-CHO or Ac-IEDT-CHO). The amount of
7-amino-4-trifluoro-methylcoumarin released was measured by
fluorometry (Fluoroskan Ascent, Dainippon Pharmaceutical, Osaka, Japan)
with 400-nm excitation and 505-nm emission filters. Data are expressed
as change in fluorescence (F) · min
1 · mg
protein
1.
Statistics. The differences between two dependent groups were evaluated with the unpaired Student's t-test. In the cases where a nonparametric test was required, data were analyzed with the Mann-Whitney U-test. Comparison among multiple groups was performed with one-way ANOVA followed by Fisher's protected least significant difference test. The results are presented as means ± SE and were considered significant when P < 0.05.
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RESULTS |
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Early release of mitochondrial cytochrome c during
ischemia.
Cytochrome c was detectable in the cytosolic fraction 15 min
after clamping. It reached a maximum level 90 min after the start of
ischemia (Fig. 1A). In
the control liver samples, cytochrome c was barely detected.
The same membrane was reprobed with a monoclonal antibody against
cytochrome oxidase subunit IV. The absence of cytochrome oxidase
subunit IV in the membrane confirmed that there was no contamination of
mitochondria in the cytosolic fraction (data not shown). Cytochrome
c in the corresponding mitochondrial fraction decreased in a
time-dependent manner during ischemia (Fig. 1B).
Reprobing the same membrane with anti-cytochrome oxidase subunit IV
antibody revealed that an equal amount of mitochondria were loaded in
each lane. There was no difference in cytochrome oxidase subunit IV
levels among time points. Figure 1C shows the sequential
changes of cytochrome c concentration in the cytosol. Cytochrome c in the cytosol increased significantly after 15 min of ischemia compared with levels in controls
(P = 0.047) and continued to increase during the
ischemic period. After 3 h of reperfusion, cytosolic
cytochrome c increased ~1.5-fold compared with the
concentration 1 h after reperfusion (P < 0.001;
Fig. 1C).
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Cytochrome c immunohistochemistry.
To evaluate the types and extent of the cells that release cytochrome
c from mitochondria, cytochrome c immunostaining
was performed. In the control liver samples, anti-cytochrome
c antibody showed punctate localization as was also the case
for the anti-cytochrome oxidase subunit IV antibody (Fig.
2, A and B). In the
liver subjected to 90 min of ischemia, hepatocytes and
sinusoidal lining cells (SLCs) lost their cytochrome c
immunoreactivity, reflecting either the release and degradation or a
conformational change of cytochrome c (2, 17).
Loss of immunoreactivity of cytochrome c was localized mainly in zones 2 and 3 (Fig. 2C). In
contrast, the immunoreactivity and localization of cytochrome oxidase
subunit IV did not change during ischemia (Fig. 2D).
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Activation of caspase-3 and cleavage of caspase substrate during
ischemia.
To assess the presence or absence of proteolysis of caspase-3, liver
homogenates were immunoblotted with anti-caspase-3 antibody. As shown
in Fig. 3, the cleaved fragment of
caspase-3 appeared 30 min after clamping. This fragment was
most evident 3 h after reperfusion. To confirm the functional
significance of caspase-3 proteolysis in ischemia and how far
apoptotic signal transduction proceeds, the samples were also
immunoblotted with anti-ICAD antibodies, which recognize the
NH2 terminus of ICAD. A 12-kDa fragment of ICAD was
observed at 30 min of ischemia, and cleaved ICAD was most
apparent 3 h after reperfusion (Fig. 3). When compared with controls, caspase-3 activity increased significantly after 90 min of
ischemia (P = 0.02), after 1 h of
reperfusion (P < 0.01), and after 3 h of
reperfusion (P < 0.01). Also, caspase-3 activity 3 h after reperfusion was significantly higher than that 1 h
of reperfusion (P = 0.04).
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Apoptosis assay.
To determine whether apoptosis was manifested during
ischemia or after reperfusion, apoptosis was assessed
with hematoxylin and eosin staining, TUNEL staining, and DNA
fragmentation. When TUNEL-positive hepatocytes and SLCs were counted
for each sample in 20 randomly chosen areas under high magnification
(×400), the number of TUNEL-positive cells in the liver subjected to
90 min of ischemia (hepatocytes 0.3 ± 0.1%, SLCs
0.6 ± 0.2%; means ± SE) was not different from that in the
control liver (hepatocytes 0.3 ± 0.1%, SLCs 0.9 ± 0.4%;
Fig. 4, A and E).
The number of TUNEL-positive cells significantly increased 1 h
after reperfusion (hepatocytes 3.4 ± 1.0%, SLCs 4.5 ± 0.5%) compared with that after 90 min of ischemia
(P = 0.04 and P = 0.03, respectively)
and markedly increased in the liver 3 h after reperfusion
(hepatocytes 18.6 ± 1.9%, SLCs 18.3 ± 2.4%;
P < 0.001 and P < 0.001, respectively; Fig. 4, B, C, and E).
Cells with morphological features of apoptosis (condensed chromatin, nuclear fragmentation, and aggregation of chromatin at the
nuclear membrane) were also observed in hematoxylin- and eosin-stained
sections 3 h after reperfusion (Fig.
5A). Furthermore, DNA
laddering was observed 1 and 3 h after reperfusion (Fig.
6). On the other hand, there was no
significant increase in TUNEL-positive cells in the liver samples
subjected to 180 min of ischemia (hepatocytes 0.38 ± 0.12%, SECs 0.73 ± 0.38%; Fig. 4, D and
E). When ischemia was prolonged up to 270 min, the
cells with apoptotic morphological changes were scarcely visible,
and most of the cells exhibited necrotic morphology (Fig.
5B).
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Western blotting of caspase-8 and Bid and measurement of casapse-8
activity.
To assess the activation of the death-receptor pathway, we performed
Western blotting of caspase-8 and Bid and a caspase-8 activity assay.
Proteolysis of procaspase-8 and Bid occurred simultaneously after
reperfusion (Fig. 7A) but not
in ischemia. Caspase-8 activity increased significantly after
reperfusion (Fig. 7B).
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TNF- in serum and tissue.
TNF-
expression was not detected in liver tissue during
ischemia with the use of Western blotting analysis (data not
shown), and there was no significant TNF-
elevation in serum during
ischemia. After reperfusion, serum TNF-
levels increased
significantly (Fig. 8).
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Inhibition of caspase activity.
The general caspase inhibitor ZVAD-fmk significantly reduced I/R injury
as indicated by serum ALT- and TUNEL-positive hepatocytes and SLCs
3 h after reperfusion compared with levels in the nontreated group
(Table 1). During the ischemia,
the caspase inhibitor did not block the release of cytochrome
c from mitochondria, although it blocked caspase-3 and -8 activities completely (P < 0.01 and P < 0.01, respectively). In contrast, 3 h after reperfusion, the general caspase inhibitor significantly suppressed cytochrome c levels in the cytosolic fraction as well as caspase-3 and
-8 activities (P = 0.03, P < 0.001, and P < 0.01, respectively; Table 1).
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DISCUSSION |
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Recent studies have shown that apoptosis plays an
important role in the pathogenesis of I/R injury in liver (7,
19). Indeed, our study also showed that caspase inhibitors block
apoptosis of hepatocytes and SLCs and reduce serum ALT levels.
Hepatic I/R injury has been reported to occur after reperfusion and to
be mediated by various factors, including reactive oxygen species and
TNF- (5, 31), which are thought to be activated by
reperfusion. However, little information exists about the type of
signaling pathway and the initiation point of apoptosis in
these models.
Previous studies have shown that cytochrome c accumulates in the cytosol in response to multiple apoptotic stimuli (18, 41) and that its release precedes morphological signs of apoptosis (3). Although mitochondrial cytochrome c release was reported in focal cerebral ischemia models (11, 12), no report has referred to changes in mitochondrial cytochrome c during liver ischemia. Our study presents the first evidence that mitochondria release cytochrome c into the cytosol during warm ischemia in the rat liver. Immunostaining of cytochrome c showed that both the hepatocytes and SLCs released cytochrome c into the cytosol and that they were localized mainly in zones 2 and 3. This might be related to the vulnerability of zones 2 and 3 to ischemic stress.
Cytochrome c with the apoptotic protease-activating factor-1-caspase-9 complex proteolytically processed and activated caspase-3 to induce apoptosis in a cell-free system (24). Our results also showed a possible involvement of mitochondria in an apoptotic pathway that is initiated during ischemia. The mitochondrial cytochrome c was released as early as 15 min after the start of ischemia and was followed at 30 min by caspase-3 and ICAD cleavage. Caspase-3 activity also significantly increased after 90 min of ischemia compared with that in controls. These results indicate that the released cytochrome c induced subsequent caspase activation and ICAD cleavage, which is further supported by the findings that the general caspase inhibitor did not inhibit the mitochondrial cytochrome c release, although it completely blocked caspase activation during ischemia. Numerous apoptotic proteases promote ICAD cleavage. Calpain cleaved ICAD into ~19- and 25-kDa fragments, and high concentrations of caspase-7 also cleaved ICAD. In physiological concentrations, only caspase-3 can cleave ICAD at the NH2-terminal caspase site to produce the 12-kDa fragment (38). Our result, the prominent 12-kDa fragment of ICAD, suggests that ICAD was cleaved by caspase-3.
Recently, Li and colleagues (23) have reported that
activated caspase-8, through death-receptor stimulation, cleaves Bid and that translocation of the truncated Bid from cytosol to
mitochondria then mediates the release of cytochrome c. The
present study revealed that there was no significant increase in
caspase-8 activity during ischemia, and caspase-8 and Bid were
first cleaved after reperfusion. Caspase-8 activation and the cleavage
of Bid after reperfusion indicate that the death-receptor pathway was
activated after reperfusion. That, together with the fact that TNF-
was detected after reperfusion, leads us to believe that TNF-
might
activate the death-receptor pathway in liver I/R injury. Furthermore,
caspase-3 was activated more after reperfusion than during
ischemia, and the general caspase inhibitor did not block
cytochrome c release during ischemia but did inhibit
its release after reperfusion. These findings suggest that the
death-receptor pathway may contribute to further increases in both
mitochondrial cytochrome c release and caspase-3
activity after reperfusion.
The activation of caspase-3 usually leads to cleavage of cytoplasmic substrates for the manifestation of apoptotic morphological changes (6, 21, 28, 37). In caspase-3-null cells, DNA fragmentation was delayed or absent (16, 34, 39, 43). ICAD cleavage at NH2-terminal caspase sites is both necessary and sufficient for CAD activation, which promotes apoptotic internucleosomal DNA fragmentation (30, 38). In this study, we did not observe apoptotic manifestations during ischemia despite caspase-3 activation and the cleavage of ICAD at NH2-terminal sites. In the 180-min ischemia model, there were neither TUNEL-positive cells nor cells with morphological manifestations of apoptosis (including nuclear shrinkage and fragmentation). In the liver samples subjected to 270 min of ischemia, most of the cells showed necrotic morphological changes. After reperfusion, however, we observed TUNEL-positive cells, DNA fragmentation, and cells with morphological manifestations of apoptosis. Although caspase-3 activation and cleavage of ICAD had already occurred during ischemia, they failed to induce biochemical or morphological changes characteristic of apoptosis during the ischemic period. This finding is further supported by a previous report that in the hypoxic, perfused liver, cell death occurred as necrosis, although caspase-3 was activated (40). Several reports have shown that apoptosis is composed of several ATP-dependent steps and that the availability of intracellular ATP determines whether cells undergo apoptosis or necrosis (8, 22). In the mitochondrial pathway, caspase-3 activation requires ATP or dATP (9). An active nuclear transport mechanism that requires ATP hydrolysis has been shown to be involved in apoptotic changes of the nuclei (42). During ischemia, intracellular ATP is rapidly exhausted as a result of insufficient oxygen supply and rapid consumption of glucose so that ischemia reduces the ATP content of liver tissue (14, 15). A study in cultured kidney cells showed that incubation of previously hypoxic cells in glucose-free medium led to cell death with necrotic morphology despite activation of the mitochondrial apoptotic cascade. In contrast, cells reoxygenated in the presence of glucose showed apoptotic morphology (29). These studies indicate that apoptosis consumes ATP.
The present results, showing that the morphological changes characteristic of apoptosis were observed only after reperfusion, indicate that whatever was generated during reoxygenation (probably ATP) might be required in order to manifest apoptosis. However, it remains unclear how a cell can produce ATP during reperfusion, because mitochondrial electron transport might be impaired due to loss of cytochrome c. One possibility is that anaerobic glycolysis may produce ATP to execute apoptosis. Another possible explanation is that, as Martinou and Green (27) recently proposed, a "mild death signal" might alter only a subpopulation of the mitochondria, and the spared mitochondria would be able to produce enough ATP to activate caspase, allowing cells to undergo apoptosis. Further studies are required to confirm the connection between ATP and apoptotic manifestation.
In summary, this study reveals that although apoptosis is manifested after reperfusion, mitochondrial cytochrome c release and caspase activation proceed during ischemia.
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ACKNOWLEDGEMENTS |
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We thank K. Matsunaga for technical advice and A. Ishida, K. Sakura, and Y. Shimojo for expert technical assistance.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Miyagawa, Shinshu Univ. School of Medicine, Asahi 3-1-1, Matsumoto 390-8621, Nagano, Japan (E-mail: shinichi{at}hsp.md.shinshu-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 6 September 2000; accepted in final form 16 May 2001.
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