Thomas E. Starzl Transplantation Institute, Departments of 1 Surgery and 2 Pathology, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania 15213
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ABSTRACT |
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The
role of NF-B, the rapid-response transcription factor for multiple
genes, in cold ischemia-reperfusion (I/R) injury was examined
after syngeneic transplantation of liver grafts. Lewis rat recipients
were killed 1-48 h after reperfusion of three different liver
grafts: 1) uninfected control, 2) infected ex
vivo with control adenoviral vector (AdEGFP), and 3)
infected ex vivo with AdI
B. In uninfected control livers, NF-
B
was activated biphasically at 1-3 and 12 h after reperfusion
with aspartate transaminase (AST) levels of 4,244 ± 691 IU/l. The
first peak of NF-
B activation associated with an increase of mRNA
for TNF-
, IL-1
, and IL-10. AdEGFP transfection resulted in
similar outcomes. Interestingly, AdI
B-transfected liver grafts
suffered more severe I/R injury (AST >9,000 IU/l). Transfected I
B
was detected in transplanted livers as early as 6 h, and this
correlated with the abrogation of the second, but not the first, peak
of NF-
B activation at 12-48 h and increased apoptosis.
Thus inhibition of the second wave of NF-
B activation in
I
B-transfected livers resulted in an increase of liver injury,
suggesting that NF-
B may have a dual role during liver I/R injury.
liver transplantation; preservation injuries; IB; adenoviral
vector
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INTRODUCTION |
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LIVER TRANSPLANTATION HAS been widely used as the therapy for patients with end-stage liver disease. However, the transplant procedure obligates cold perfusion, hypothermic storage, warm ischemia, and warm reperfusion of the liver grafts, resulting in ischemia-reperfusion (I/R) injury of the transplanted liver. Although the introduction of University of Wisconsin (UW) solution has significantly improved clinical outcome, I/R injury remains one of the major clinical problems after liver transplantation. Prolonged cold ischemic storage, as well as poor donor quality such as hepatic steatosis, increase I/R injury, resulting in dysfunction or nonfunction of the transplanted liver grafts (1, 16, 37, 39, 43).
Multiple factors have been shown to contribute to I/R injury. The lack of oxygen during ischemia induces depletion of ATP followed by a deterioration of the intracellular Ca+ and Na+ homeostasis and the activation of cytotoxic enzymes (e.g., proteases, phospholipases, endonucleases). Additionally, generation of reactive oxygen species during reperfusion is believed to contribute to direct injury of the cells in the liver. Accordingly, endothelial cell damage and disturbance of microcirculation and Kupffer cell activation initiate the complex network of proinflammatory signal-transduction cascade. Subsequent recruitment of neutrophils further amplifies tissue damage (12, 15, 25).
NF-B, an inducible dimeric transcription factor that belongs to the
Rel family of transcription factors, is a main mediator of the cellular
response to a variety of extracellular stress stimuli. The targets for
transcriptional activation of NF-
B include the genes for cytokines,
acute phase-response proteins, immunoglobulins, and cell-adhesion
molecules (30). It has been shown that NF-
B is
activated during the acute phase of I/R injury after liver transplantation (7, 19). However, the role of NF-
B
activation during I/R injury of the transplanted liver is not clear. As
a proinflammatory transcription factor, NF-
B may trigger
upregulation of cytokines (e.g., TNF-
and IL-1) and adhesion
molecules (e.g., ICAM-1), thereby initiating inflammatory responses.
However, NF-
B also plays a role in acute cellular stress responses
such as in the protection from TNF-
-induced apoptosis
(3, 44). NF-
B has been shown to be an essential
survival factor in several physiological conditions including embryonal
liver development and liver regeneration (4, 24). Thus
NF-
B possibly induces both harmful (inflammatory) and beneficial
(antiapoptotic/survival) effects during I/R injury.
Activation of NF-B is tightly controlled by its endogenous inhibitor
I
B, which complexes NF-
B in the cytoplasm. Phosphorylation and
proteolytic degradation of I
B allows the release and nuclear translocation of NF-
B, followed by transcription of many
proinflammatory genes. Degradation of I
B is linked to
phosphorylation of serine residues 32 and 36 located in the
NH2-terminal part of the polypeptide. A mutant form of
I
B
, in which serines 32 and 36 are replaced by alanine residues
(S32A/S36A), has been shown to be effectively protected from
degradation and thereby prevents NF-
B activation (45).
This study was undertaken to examine the detailed activation pattern of
NF-B in I/R injury induced in rat liver grafts stored in UW solution
for 18 h and subsequently transplanted in syngeneic recipients. In
addition, we attempted to evaluate the role of NF-
B in this animal
model by overexpressing a dominant-negative mutant form of I
B
(S32A/S36A) using the adenoviral gene-transfer technique (11,
41).
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MATERIALS AND METHODS |
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Viral vectors.
The adenovirus encoding the IB
superrepressor gene
(AdI
B) was kindly provided by D. A. Brenner
(University of North Carolina) (24, 27, 42). The cDNA
insert of plasmid pRc/CMV-I
B
S32A/S36A, which contains a
hemagglutinin (HA)-tagged human superrepressor of NF-
B, was
subcloned into the Xbal site of the pACCMV.PLPASR(+) plasmid
to construct the plasmid pACCMV/I
B, in which I
B
is driven by
the cytomegalovirus (CMV) promoter/enhancer. The adenovirus encoding the enhanced green fluorescent protein gene under a CMV promoter (AdEGFP) was used as a control (17, 33, 41).
Orthotopic liver transplantation and viral vector delivery to liver grafts. Inbred male Lewis rats (Lew: RT1l, Harlan Sprague Dawley, Indianapolis, IN) were used for all syngeneic transplantation experiments in this study to prevent immunological interference. Rats weighing 200-300 g were maintained in a laminar-flow, specific-pathogen-free atmosphere at the University of Pittsburgh. Liver graft harvesting and orthotopic transplantation without hepatic arterial reconstruction were performed as described previously by Kamada and Calne (28). Adenoviral vector was delivered to the liver grafts by ex vivo cold infusion into the harvested liver via the portal vein and hepatic artery with clamp technique (CT) (11, 41). Briefly, the donor liver was flushed in situ with 20 ml of cold (4°C) UW solution (DuPont, Merck Pharmaceuticals, Wilmington, DE) through the infrarenal aorta. After a routine skeletonization, the isolated liver graft was slowly perfused with a total of 6 ml UW containing adenoviral vector (1 × 109 pfu); after the initial flush out with 2 ml via the portal vein and 1 ml via the hepatic artery, vascular clamps were placed on the supra- and infrahepatic inferior vena cava, and subsequent perfusate via the portal vein (2 ml) and hepatic artery (1 ml) was infused into the liver, retaining the viral suspension in the hepatic vasculature. After visible expansion of the liver capsule was noted, the hepatic artery was ligated and the portal vein was clamped. Liver grafts were stored in UW solution at 4°C for a total preservation period of 18 h. Stored livers were transplanted into syngeneic Lew recipients after being rinsed with 3 ml of cold lactated Ringer solution via the portal vein. Chia et al. (11) and Takahashi et al. (41) previously showed that adenoviral gene delivery with CT induced 10-30% infectivity exclusive to hepatocytes using a dose of 1 × 109 pfu.
Recipient rats were killed at 1, 3, 6, 12, 24, and 48 h after transplantation (n = 2-5 for each time point). Blood samples were collected from the aorta, and the liver grafts were harvested for further studies. All procedures in this experiment were performed according to the guidelines of the Council on Animal Care at the University of Pittsburgh and the National Research Council's Guide for the Care and Use of Laboratory Animals.Isolation of nuclear and cytoplasmic proteins.
Frozen liver tissues were suspended in buffer containing 10 mM Tris (pH
7.5), 1.5 mM MgCl2, 10 mM KCl, and 0.1% Triton X-100 and
lysed by douse homogenization. Nuclei were recovered by
microcentrifugation at 7,500 rpm for 5 min. The supernatant containing
cytoplasmic and membrane protein was collected and stored at 80°C
for Western blot analysis. Nuclear proteins were extracted at 4°C by
gently resuspending the nuclei pallet in buffer containing 20 mM Tris (pH 7.5), 20% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM
EDTA, and 0.1% Triton X-100, followed by 1 h incubation at 4°C
with occasional vortexing. After microcentrifugation at 13,000 rpm for
15 min at 4°C, the supernatant containing nuclear protein was
collected and frozen at
80°C for EMSA. All buffers contained the following additional ingredients: 0.2 mM phenylmethylsulfonyl fluoride, 0.5 mM ditiothreitol, and 0.1 mM Na vanadate. Protein concentration was quantitated with Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA).
Western blot analysis.
To detect the transfected IB
superrepressor expression in liver
grafts, Western blot analysis was carried out using cytosolic proteins.
One hundred micrograms of cytosolic proteins were separated by
electrophoresis on 10% acrylamide sodium dodecyl surface gels and
transferred to nitrocellulose membranes (Scleicher and Schuell, Keene,
NH). For the blocking of nonspecific binding, 5% nonfat dry milk in
PBS-Tween was added to the membrane for 2 h at room temperature.
Membranes were washed in PBS-Tween and then incubated with primary
rabbit polyclonal anti-HA antibody (Y-11, 1:1,000, Santa Cruz
Biotechnology, Santa Cruz, CA) for 1.5-2 h. After repeat washings
with PBS-Tween, membranes were incubated with secondary goat
anti-rabbit antibody (1:10,000, Pierce Chemical, Rockford, IL) for 45 min. Membranes were developed with the SuperSignal detection systems
(Pierce Chemical) and exposed to film.
EMSA.
NF-B DNA binding activity was measured by EMSA using nuclear
extracts from graft liver tissues. The NF-
B oligonucleotide (Life
Technologies, Rockwell, MD) was based on the NF-
B sequence in the
immunoglobulin light-chain enhancer. DNA probes were prepared by end
labeling with [
-32P]dATP (DuPont, Merck
Pharmaceuticals) and T4 polynucleotide kinase (Boehringer-Mannheim
Biomedical Products, Mannheim, Germany) and purified in Tris-EDTA
buffer containing NaCl (100 mM) using G-50 resin columns
(Whatman, Newton, MA). Typically, 5 µl (10-20 µg) of hepatic
nuclear extract were incubated with ~100,000 counts/min of
32P-labeled oligonucleotides (~0.5 ng) for 1-2 h at
room temperature in a buffer containing 10 mM Tris (pH 7.6), 10%
glycerol, 1 mM EDTA, 1 mg/ml BSA, and 0.2% Nonidet P-40. Additionally,
2-4 µg of poly(dI-dC) (Boehringer-Mannheim Biomedical
Products) were added to the mixture as nonspecific competitor DNA.
Protein-DNA complexes were resolved on 4% nondenaturing polyacrylamide
gels in 0.4 × running buffer containing 450 mM Tris borate and 1 µM EDTA (pH 8.0). Gels were dried after electrophoresis and subjected to autoradiography.
RNA preparation and RNase protection assay. Total RNA was extracted from frozen samples by using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. The concentration of RNA was determined by ultraviolet (UV) spectrophotometer at 260 nm.
RNase protection assay (RPA) using commercially available kits (all from PharMingen, San Diego, CA) was used to quantify mRNA levels for cytokines and apoptosis-related molecules. Radiolabeled antisense RNA multiple probes were synthesized using In Vitro Transcription kit and rat cytokine multiprobe template sets (rCK-1 and rAPO-1, PharMingen), which included probes for cytokines (IL-1Liver function tests. Hepatic function and injury following rat liver transplantation was assessed by serum aspartate aminotransferase (AST) and serum alanine aminotransferase (ALT) levels using the Opera Clinical Chemistry System (Bayer, Tary Town, NY).
Routine histopathology. Liver graft tissues were fixed in 10% formalin, embedded in paraffin, sectioned into 6-µm thickness, and stained with hematoxylin and eosin. Severity of I/R injury was evaluated based on the degree of sinusoidal congestion, neutrophil infiltration, and necrosis of parenchymal cells. Samples were semiquantitively graded on the scale of 0-4 as none, minimal, mild, moderate, or severe, by the pathologist who was unaware of the treatment groups.
Apoptosis.
Liver tissue was fixed with optimum cutting temperature compound and
cut to 6-µm-thick sections. The sections were fixed in cold methanol
for 20 min and then air dried for 20 min. After the sections were
washed in PBS, they were incubated with the TUNEL mixture kit
(Boehringer- Mannheim) for 40 min at 37°C, then with secondary
antibody streptavidin 488 (Molecular Probes, Eugene, OR) for 40 min.
Sections were further assessed using anticleaved caspase-3 antibody
(Cell Signaling Technology, Beverly, MA) that detects caspase-3
activation, a final step of the caspase cascade. Sections were viewed
using a Provis upright fluorescent microscope, and the number of
positively stained cells was determined by blindly counting the number
of labeled cells in 20 low-power field (×20) per section. Pictures
were taken with a Nikon high-resolution digital camera.
DNA fragmentation assay.
Liver tissue was homogenized using a dounce homogenizer and an organic
extraction of DNA was performed using a 1:1 suspension of
phenol and chloroform. After 1/10 volume of 3 M Na acetate (pH 5.2) was
added to the extraction, the DNA was precipitated by adding an equal
volume of cold isopropanol and stored overnight at 20°C. The DNA
pellet was then washed twice with 75% ethanol and resuspended in
buffer containing 10 mM Tris · HCl (pH 8.0) and 1 mM
EDTA. After RNA were digested with RNase at 37°C for 2 h,
samples (30 µl) were mixed with gel-loading buffer [0.05% (wt/vol)
bromophenol blue, 40% (wt/vol) sucrose, 0.1 M EDTA (pH 8.0)] and
electrophoresed through a 1.2% agarose gel. Gels were stained with
ethidium bromide and visualized by UV transillumination. A
double-stranded 123-bp DNA ladder served as a standard.
Statistical analysis. Data are represented as the mean ± SE. Comparisons between the groups at different time points after reperfusion were performed using the Student's t-test or ANOVA using the StatView program (Abacus Concepts, Berkeley, CA). Differences were considered significant at P values <0.05.
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RESULTS |
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NF-B DNA binding activity following cold I/R.
After the liver graft underwent 18 h of cold preservation and
reperfusion, NF-
B DNA binding activity in the liver markedly rose
(Fig. 1A). Sequential analysis
after I/R showed that NF-
B DNA binding activity started to increase
at 20 min (data not shown), significantly increased at 1-3 h,
and dropped at 6 h after reperfusion. This early peak of NF-
B
binding activity at 1-3 h contained two distinct protein-DNA
complexes. Subsequently, the second peak of NF-
B binding activity
was observed at 12 h after reperfusion, which diminished by
24-48 h.
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mRNA for cytokines and apoptosis-associated molecules
following cold I/R.
Early after cold I/R injury, there was a sharp increase of mRNA for
cytokines (Fig. 2A). mRNA for
IL-1, -1
, -6, and -10 and TNF-
increased 5- to 10-fold at
1-3 h after I/R, gradually decreased, and returned to baseline
level by 12 h. IL-6 and -10 and TNF-
were downregulated
thereafter, whereas IL-1
and -1
showed a secondary small increase
48 h after I/R. As expected, Th1-type cytokines, such as IL-2 and
IFN-
(not shown), were not involved in the early phase of I/R
injury.
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AdIB expression by adenovirus-mediated gene therapy using clamp
technique.
We have previously shown that the back-table CT using AdLacZ and AdEGFP
provides a highly efficient gene transfer to liver grafts (11,
41). AdI
B, which contains an HA-tagged human superrepressor
of NF-
B (1 × 109 pfu), was delivered to the liver
grafts with CT, and early time course of transferred gene expression
was studied by Western blot analysis using anti-HA antibody (Fig.
3). Normal liver without transplantation
was negative. Transfected I
B gene product started to
appear at 6 h after reperfusion, increased with time, and was maintained at a high level for 48 h after reperfusion.
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Effects of AdIB gene therapy on NF-
B DNA binding activity.
The effects of AdI
B or AdEGFP infection on NF-
B activation were
evaluated by EMSA using liver nuclear extracts obtained at different
time points after reperfusion (Fig. 4).
The early peak of NF-
B at 1-3 h was not affected by
AdI
B or AdEGFP gene transfer, and potent DNA
binding activity was seen in all groups of liver grafts early after
reperfusion. In contrast, NF-
B DNA binding activity was partially
inhibited in AdI
B-treated liver grafts between 12 and 48 h,
compared with AdEGFP-treated or control liver grafts. The inhibition of
the second, but not the first, peak of NF-
B DNA binding correlated
with the timing of transferred I
B protein expression (Fig. 3).
Zwacka et al. (49) showed dissociation of NF
B-I
B is
induced by tyrosine phosphorylation of I
B rather than proteolytic
degradation of I
B in the mouse hepatic warm ischemia model.
In this study, we did not directly address the relative importance of
tyrosine phosphorylation, and it is possible that the early 1-h NF-
B
peak rate is explained by this mechanism. However, the fact that the
superrepressor AdI
B did diminish NF-
B binding activity at
12-48 h suggests that serine phosphorylation is a triggering
event.
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Effects of AdIB gene therapy for liver injury.
The effects of AdI
B gene transfer on the hepatic cold I/R
injury were examined by measuring in the liver transaminase (serum AST
and ALT) and by histopathology. Both AST and ALT in control group
increased soon after reperfusion, reaching peak levels (4,244 ± 691 U/l) at 24 h with a subsequent gradual decrease (Fig.
5). Most interestingly, AST and ALT
significantly increased in recipients of AdI
B-treated liver grafts,
compared with control and AdEGFP-treated animals. Peak values at
24 h after reperfusion were doubled in AdI
B-infected liver
recipients, and both AST and ALT were still significantly high at
48 h (P < 0.05).
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DISCUSSION |
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This study shows a biphasic pattern of NF-B activation in cold
I/R injury of liver grafts. The early peak of NF-
B DNA binding was
seen 0.5-3 h after reperfusion and represents the nuclear translocation of NF-
B p50/p65 heterodimer and possibly p50
homodimers. NF-
B DNA binding activity subsequently diminished for a
few hours, and then a second peak of nuclear DNA binding was observed
at 9-12 h after reperfusion. The second peak of NF-
B activation was less vigorous than the first and was mainly composed of p50 homodimer. The timing of the early peak of NF-
B activation
correlated well with the acute phase of I/R injury. During the acute
phase (~6 h after reperfusion), the burst of reactive oxygen
intermediates (peroxide, superoxide, and hydroxyl radicals) generated
by I/R induces cellular damage by means of protein
oxidation/degradation, lipid peroxidation, and DNA damage. Subsequent
cellular responses to the damage include the activation of
proinflammatory signal-transduction pathways, changes in the
microcirculation and interactions between leukocytes, cytokine
production, and induction of chemoattractants. These changes are
believed to initiate the subacute phase of I/R injury that is
characterized by massive neutrophil infiltration, peaking 18-24 h
after reperfusion.
Early activation of NF-B after I/R, similar to the first peak of
NF-
B activation seen in this study, has been well documented in both
warm (23, 40, 49) and cold (6, 7, 19) I/R injury. In an effort to examine the role of NF-
B activation after I/R, several studies attempted to inhibit early NF-
B binding activity using antioxidant/anti-NF-
B reagents (e.g.,
diethyldithiocarbamate) (9, 23, 31, 32), double-stranded
DNA with a specific affinity for NF-
B (35), or I
B
superrepressor (6). Inhibition of early NF-
B binding
activity in the nucleus was found to associate with downregulation of
proinflammatory cytokines (e.g., IL-1
and -6 and TNF-
) and
inducible nitric oxide synthase with an amelioration of I/R-induced
tissue injuries (7, 9, 23, 32, 35). These results identify
the role for NF-
B in the early cascade of I/R injury: I/R releases
free radicals that induce cellular damage, followed by NF-
B
activation, which, in turn, initiates proinflammatory
signal-transduction pathways. Thus early activation of NF-
B in I/R
injury seems to have harmful (inflammatory) effects, and therefore,
inhibition of early NF-
B activation after I/R is found to be protective.
On the other hand, the second peak of NF-B activation at 9-12 h
after reperfusion has not received much attention. Chandrasekar and
Freeman (8) and Chandrasekar et al. (9)
showed a biphasic regulation of NF-
B at 15 min and 3 h in rat
hearts after 15 min of warm I/R. Lille et al. (32) also
demonstrated a biphasic activation (0.5-3 and 6-16 h) of
NF-
B in I/R of rat skeletal muscle following a 4-h warm
ischemia. However, little is known about the cause and role of
the second peak of NF-
B activation. We have found in this study that
the delivery of AdI
B partially inhibited the second peak of NF-
B
activation and resulted in dramatic worsening of the hepatic I/R
injury, suggesting that the later phase of NF-
B activation at
9-12 h after reperfusion may relate to beneficial effects. In
contrast to the early NF-
B activation, the second peak of
NF-
B was not associated with subsequent proinflammatory
cytokine upregulation, supporting a nonproinflammatory role for the
late phase of NF-
B activation (9).
Definite roles of secondary NF-B activation in I/R injury need to be
further investigated; however, on the basis of several observations,
some hypotheses can be illustrated. A possible discrete role of
secondary NF-
B activation in this study may relate to the fact that
the second peak is mainly composed with NF-
B p50 homodimer. The
NF-
B transcription factor family consists of five different members:
p50, p52, p65 (RelA), c-Rel, and RelB and various forms of homo- and
heterodimers. The induced NF-
B prototype in many cell types is
predominantly composed of the p50/p65 heterodimer subunit, which
possesses strong transactivating potential as a transcription factor in
the NF-
B family. As shown in hepatic warm I/R injury (23, 40,
49), the early peak of NF-
B activation after cold I/R in this
study contained NF-
B complexes of p50/p60 heterodimers. In contrast,
the secondary phase of NF-
B activation in this study was composed of
p50 homodimers, which may downregulate NF-
B-mediated initial
responses to subsequent stimuli in the regulatory step. Because p50
homodimer lacks transcription activation domains, nuclear translocation
of p50 homodimer may inhibit NF-
B-mediated transcription by
competing with other NF-
B complexes for access to binding sites
(48, 49). Inhibition of p50 homodimers with AdI
B in
this study may result in persistent transcription of proinflammatory
genes. However, our preliminary data show that AdI
B treatment does
not significantly alter mRNA levels of proinflammatory cytokines (data
not shown), suggesting that secondary NF-
B activation may have
different roles.
NF-B has been suggested to play an essential role in cell
proliferation and differentiation. NF-
B controls the expression of a
number of growth-promoting cytokines. Moreover, NF-
B transcriptional activity appears to be linked to extracellular signaling that controls
cell-cycle progression. Mice lacking the p65 (RelA) subunit of NF-
B
as a result of targeted mutation of the relA gene die embryonically at 15-16 days of gestation with massive degeneration of liver cells caused by extensive apoptosis (4).
Typically, the control of mammalian cell proliferation by extracellular
signals takes place in middle to late G1 phase of the cell
cycle, and several reports have described an association of NF-
B
activation with the early G1 phase of the cell cycle
(2, 13, 14). In the liver, a rapid NF-
B DNA binding
activity is seen following partial hepatectomy, when hepatocytes
progress in the cell cycle from G0 to G1
(13). Inhibition of NF-
B activation after partial hepatectomy with AdI
B leads to massive apoptosis of
hepatocytes and liver dysfunction (24). Thus inhibition of
NF-
B correlates with failure to progress through the normal cell
cycle. It has been recently shown that NF-
B can activate the
transcription of cyclin D1, which is a key promoter for
G1-to-S phase progression (20, 21).
The recovery of the liver grafts from cold I/R injury requires liver
regeneration to replace damaged hepatic mass. Apoptosis seen in
the injured area of AdIB-transfected liver in this study is
reminiscent of that seen in the degenerating liver of NF-
B p65
knockout mice and in AdI
B-transfected liver after partial hepatectomy (4, 24). It is tempting to speculate that the inhibition of NF-
B by AdI
B results in the failure to transmit growth signals that are required for the recovery from I/R injury.
In addition to the roles on regulation of cell cycle, NF-B may
regulate apoptosis by directly controlling genes that inhibit or promote apoptosis. Apoptosis has been known to play
a role in I/R injury (18, 29, 34, 47), and inhibition of
apoptotic signals by administration of caspase-3 inhibitor
(36), or by overexpression of antiapoptotic Bcl-2,
ameliorates I/R injury (5). The antiapoptotic function
of NF-
B is further supported by several studies demonstrating that
NF-
B activity prevents the induction of apoptosis by
TNF-
, ionizing radiation, and anticancer agents (3, 44,
45). Candidates of target genes include caspase-8 and -3 (22) and antiapoptotic molecules, such as cellular inhibitors of apoptosis (10, 38, 46). It is
therefore considered a possibility that increased I/R injury in
AdI
B-infected liver grafts is due, in part, to the inhibition of the
antiapoptotic functions of NF-
B. Together, these findings
suggest a complex balance for NF-
B in regulating proinflammatory
cytokine gene expression, cell proliferation/differentiation, and
apoptosis-related signaling during the reperfusion period
following liver transplantation.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. C. Watkins and M. Tabacek for technical help and C. Forsythe for preparing the manuscript.
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FOOTNOTES |
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This work was supported by NIH Grants R01AI-138899 and R01GM-52021 and the George H. A. Clowes, Jr. Memorial Research Career Development Award of the American College of Surgeons (to D. A. Geller).
Address for reprint requests and other correspondence: N. Murase, Thomas E. Starzl Transplantation Institute, Univ. of Pittsburgh, 200 Lothrop St., Pittsburgh, PA 15213 (E-mail: murase+{at}pitt.edu).
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.
July 17, 2002;10.1152/ajpgi.00515.2001
Received 7 December 2001; accepted in final form 9 July 2002.
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