1 Institut National de la
Santé et de la Recherche Médicale Unité 519 and
Institut Fédératif de Recherches Multidisciplinaires sur
les Peptides, Cirrhotic liver is considered to regenerate less
actively than normal liver after hepatic resection. However, the
mechanisms responsible for this impaired regeneration and the cross
talk of implicated factors still remain unclear. In the present study, mRNA levels for cyclins, growth factors, and cytokines were
quantitatively assessed by a RT-PCR method at different times after
hepatectomy in order to determine the relationships between these
factors and the impaired regenerative process observed in cirrhotic
liver. In our model of
CCl4-induced cirrhosis, mRNA
levels for cyclins and thymidine kinase provide evidence for the
impaired and delayed hepatic regeneration. Moreover, we observed a
significant decrease in interleukin (IL)-6 and tumor necrosis
factor-
liver regeneration; c-Met; hepatocyte growth factor
LIVER SURGERY IS LARGELY used in humans as a
consequence of the increased frequency of primary hepatocellular
carcinoma (HCC) particularly in post-hepatitis C cirrhosis. Surgical
resection is a potentially curative therapy, but the outcome of a
radical hepatic resection is usually very poor, with a significant risk of liver failure (9). The postoperative recovery depends on the
regenerative ability of the remaining liver. Previous studies (3, 18)
have reported conflicting data regarding liver ability to regenerate
after partial hepatectomy (PH) in animals and humans with cirrhosis.
Recently, it has been reported (2, 21) that in rats with hepatic damage
liver regeneration after resection did occur but was impaired and
delayed compared with that observed in healthy animals. However, the
mechanisms responsible for this impaired regeneration and the cross
talk of the implicated factors still remain unclear.
Liver regeneration after PH is defined as an orchestrated response that
is induced by specific stimuli, and it involves numerous and sequential
changes in gene expression (8, 20). Many growth factors and cytokines,
particularly hepatocyte growth factor (HGF) (23), transforming growth
factor (TGF)- The aim of the present study was to quantify in rat mRNA levels for
growth factors (TGF- Animals.
Adult male Sprague-Dawley rats (Charles River), weighing ~200 g were
kept under a 12:12-h light-dark cycle and fed with standard laboratory
chow and water ad libitum. They were kept at rest for at least 1 wk
before the start of these studies. All animal manipulations were
performed according to the recommendations of a local ethical committee
and under the supervision of authorized investigators.
CCl4-induced cirrhosis.
Cirrhosis was induced according to a protocol modified from Proctor and
Chatamra (27). Phenobarbital at a concentration of 35 mg/dl was added
to the drinking water because it has been shown to increase liver
sensitivity to CCl4 by increasing
the activity of cytochrome P-450.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
mRNA and a significant increase for IL-1
mRNA. No
significant change of hepatocyte growth factor (HGF) mRNA level was
detected, contrasting with the decrease both at mRNA and protein levels
in the expression of the c-Met/HGF receptor. Therefore,
the impaired regeneration of the cirrhotic liver is associated not only
with a lowered level of signals that normally promote liver growth but
also with a strong decrease in c-Met receptor despite a normal
expression of its specific ligand.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(19, 30) and -
(28), interleukin (IL)-6, and tumor
necrosis factor (TNF)-
(20) appear to play important roles in this
process. Previous investigations have revealed that HGF is the most
potent mitogen for normal hepatocytes, with early increased mRNA and
plasma levels after liver injury and PH. Moreover, HGF elicits its
biological effects through its binding to a specific receptor known as
c-Met (36). It is also well established that in liver after various injurious processes cytokines are important components of the early
signaling pathways leading to specific responses in neighboring or more
distant target cells (10, 31). A recent study (25) has indicated the
importance of these factors in the pathogenesis and progression of
chronic liver diseases, with a significant activation of cytokines at
the mRNA level in end-stage cirrhosis.
, HGF, and its receptor) and proinflammatory cytokines (IL-1
, IL-6 and TNF-
) to determine the relationships between these factors and to understand how they may account for the
impaired regenerative process observed in cirrhotic liver.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Surgical procedures. Rats were anesthetized with ether, and a two-thirds hepatectomy was performed after midline laparotomy according to the method of Higgins and Anderson (12). PH was performed by excision of the median and left lateral lobes of the liver. A biopsy specimen from the resected liver lobe was stored in a 10% buffered formaldehyde for subsequent histological examination.
Two experimental groups were studied. In group 1, healthy rats were submitted to a two-thirds hepatectomy. In group 2, PH was performed after 16 wk of CCl4-induced cirrhosis. In both groups, four rats were killed at 6 h, 12 h, day 1, day 2, day 3, or day 6 after surgery. The liver was removed, one sample was stored atHistopathological studies. Macroscopic appearance and microscopic examination of liver sections verified the presence of fibrosis and cirrhosis in rats treated with CCl4-phenobarbital. After routine paraffin wax processing, trichrome blue-stained sections were prepared and the degree of liver damage was evaluated.
The biopsy specimens were examined by a liver pathologist who analyzed four main areas of fibrosis deposits as previously described by Chevallier et al. (4): centrilobular vein (CLV), perisinusoidal space (PS), portal tract (PT), and septa, taking into account the number (NS) and width (WS) of the septa. The histological score is detailed as follows: CLV, 0 = normal, 1 = moderately thickened, 2 = markedly thickened (annular aspect of vein wall with fibrosis extensions between hepatocytes); PS, 0 = normal, 1 = localized fibrosis, 2 = diffuse fibrosis; PT, 0 = normal, 1 = enlarged without septa, 2 = enlarged with septa, 3 = cirrhosis; NS, 0 = absence, 1 = <6 septa/10 mm, 2 = >6 septa/10 mm, 3 = nodular organization; WS, 1 = thin and/or incomplete, 2 = thick and loose connective matrix, 3 = very thick and dense connective matrix. Fibrosis was graded on a scale of 0 to 2. Total score of hepatic fibrosis (SHF) was calculated according to
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Preparation of NPC. Heparin (500 UI; Laboratoire Leo, Paris) in 1 ml of 0.9% saline solution was injected into the saphenous vein. The portal vein was then cannulated with a catheter, and the hepatic artery was ligated. The liver was immediately perfused with 10 ml of Gey's balanced salt solution (GBSS), excised, and removed from the peritoneal cavity. It was then transferred into a perfusion chamber to be continuously perfused according to a protocol modified from Knook et al. (15). In brief, the liver was perfused for 7 min with pronase 0.02% in GBSS buffer and next for 20 min with pronase 0.2% and collagenase (Sigma) 0.02% in GBSS/HEPES buffer. The liver was mechanically dissociated, and the liver homogenate was incubated with pronase 0.03% and collagenase 0.03% in GBSS/HEPES buffer for 45 min at 280 rpm with orbital stirring. Some micrograms of DNase I (Boehringer) were added to prevent cell clumping. The suspension was centrifuged at 600 g at 4°C for 10 min. The supernatant was discarded, and the pellet was submitted to a metrizamide (Sigma) gradient (17.5%) to separate the NPC from the nonviable parenchymal cells and erythrocytes. The low-density fraction was collected, resuspended in 20 ml GBSS, and centrifuged at 600 g for 10 min. The final pellet contained ~0.5-1.10 ×108 NPC.
Quantitative RT-PCR.
Total RNA was extracted from a frozen liver sample or from NPC
according to a one-step method. Briefly, cells were lysed with a
denaturing solution containing 4 M guanidium thiocyanate, 25 mM sodium
citrate, pH 7.0, 0.5% sarcosyl, and 100 mM 2-mercaptoethanol. Total
cellular RNA was obtained by sodium acetate-phenol-chloroform extraction, and RNA concentration was measured by spectrophotometry at
260 nm. The RNA preparations were controlled by agarose minigel electrophoresis with visualization of 18S and 28S ribosomal RNA bands
after ethidium bromide staining. Two micrograms of total RNA from each
sample were reverse-transcribed in 60 µl containing 12 µl of
5× buffer, 6 µl dNTP (10 mM each), 120 units RNasin (Promega), 400 units of Moloney murine leukemia virus RT (Promega), and 500 pmol
random hexamer primers (Pharmacia). The mixture was incubated for 60 min at 37°C, followed by heating at 95°C for 5 min. Ten microliters of the RT product were then used for PCR amplification. They were brought to a final volume of 50 µl containing 5 µl of 10× PCR buffer, 1 µl dNTP (10 mM each), 5 µl
MgCl2 (25 mM), 1.25 units
Taq polymerase (Promega), 1 µCi
[-33P]dATP, and 100 pmol of 3'- and 5'-specific primers (Eurogentec).
|
SDS-PAGE and Western blot analysis. Frozen tissues were sliced very thinly and homogenized in lysis buffer with freshly added inhibitors. Lysates were clarified by centrifugation at 12,000 g for 20 min at 4°C. The supernatants were collected, and a protein assay was performed with the use of a Bio-Rad protein assay kit (Bio-Rad Laboratories). Twenty micrograms of total protein from each sample were loaded and separated onto 8% SDS-polyacrylamide gels. After electrophoresis, proteins were transferred to an Optitran nitrocellulose membrane (Schleicher & Schuell, Céra-labo, France) and analyzed by immunoblotting. Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline containing 0.05% Tween 20 and were incubated for 1 h with biotinylated anti-c-Met rabbit polyclonal antibody (Santa Cruz Biotechnology, Tebu, France). The sheets were then incubated with the complex avidine-biotinylated peroxydase (Vector Laboratories, Coger, France) Immunoreactive bands were visualized by the enhanced chemiluminescence method (Amersham Pharmacia Biotech) and exposed to X-ray film. The expression level of MET protein in each sample was estimated with the use of densitometric scanning with Biocom equipment composed of a CCD camera (Canon) and Lecphor software.
Statistical methods. Final results are presented as means ± SD and were compared with Student's t-test. P < 0.05 was considered statistically significant.
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RESULTS |
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Survival rate. The survival rate of the CCl4-treated rats was ~80% over the first 6 wk. Deaths were probably due to acute CCl4 toxicity. Furthermore, we observed a strong decrease in body weight (data not shown) in all surviving animals with cirrhosis. Therefore, 32 rats underwent PH after a CCl4-induced cirrhosis, and we noted 8 vs. 0 deaths in cirrhotic animals vs. healthy controls.
Histological findings. The condition of liver tissue was examined in all treated rats before PH. Macroscopically, the livers were inelastic and nodular. Microscopically, SHF was evaluated as described in MATERIALS AND METHODS.
In control animals, no abnormalities were seen (Fig. 1A) and the SHF was 0. A SHF between 2 and 4 revealed a moderate fibrosis of CLV and PT (Fig. 1B). Between 6 and 10, it corresponded to a strong fibrosis with a cirrhosis not fully developed (Fig. 1C) and at 10 the cirrhosis was complete, with regenerative nodules surrounded by broad strands of fibrous tissue (Fig. 1D).
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|
Markers of the cell-cycle progression as control of the impaired
regeneration after PH in cirrhotic rats.
The mRNA levels for cyclins B, D1, and E (Fig.
2) were determined in rat liver after a PH
that was either preceded or not by a
CCl4 treatment. Before
hepatectomy, cyclins B, D1, and E were barely detectable in normal and
cirrhotic rats. In normal rats, a strong induction of the all-cyclin
mRNAs began at 12 h and peaked at day
1 posthepatectomy, whereas in cirrhotic rats a
significant decrease in cyclin B, D1, and E mRNA levels was observed at
6 h, 12 h, and day 1, followed by a
delayed peak at day 2. The difference
was most pronounced for cyclin E, whose mRNA levels were significantly
decreased in early times with a delayed peak at day
3. Interestingly, for thymidine kinase, a key
regulatory enzyme of DNA synthesis, we observed in normal rats a mRNA
level peaking at day 1 posthepatectomy, whereas in CCl4-treated rats this level was
decreased, with a delayed peak at day
3 postresection.
|
Expression of growth factors during liver regeneration after PH in
normal or cirrhotic rats.
After PH, in NPC, HGF mRNA increased as soon as 6 h, remained high at
12 h and day 1, and then declined to
the basal value at days 2-6. In
CCl4-treated rats, we did not
observe any significant difference for the expression of this growth
factor (Fig.
3A).
|
Expression of c-Met during liver regeneration after PH in normal or
cirrhotic rats.
To support the results observed at the mRNA level, we examined the
expression of the c-Met receptor at the protein level by Western blot
analysis with an antibody directed against the COOH terminus of c-Met
p140 of mouse origin. As shown in Fig. 4,
variable levels of 140-kDa c-Met protein were detected after
hepatectomies performed at different times on healthy or cirrhotic
rats. After CCl4 treatment, this
protein level decreased at early times after surgery compared with
levels observed after hepatectomy on healthy rats.
|
Cytokine expression in NPC during liver regeneration after PH in
cirrhotic rats.
In control rats after hepatectomy, the mRNA levels for the
proinflammatory cytokines increased, with a peak at 12 h for IL-1 (Fig.
5A)
and IL-6 (Fig. 5B) and with a peak
at 6 h for TNF-
(Fig. 5C), and
then gradually decreased to the preoperative level at
day 3 after surgery. In the
CCl4-treated animals, the mRNA levels of these three cytokine mRNAs were significantly lowered in the
early times (6 h and 12 h posthepatectomy) with a delayed peak at day 1 for IL-1
and TNF-
and day 3 for IL-6. However, in the
CCl4-treated rats IL-1
mRNA
levels remained strongly elevated at days
2 and 3.
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DISCUSSION |
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To understand the mechanisms involved in cirrhotic liver regeneration after PH and control thereof by growth factors and cytokines, we have analyzed the mRNA levels of some of these mediators at different time points after surgery in rat with CCl4-induced cirrhosis.
Hepatotoxic agents commonly used for induction of liver fibrosis and cirrhosis include CCl4, dimethylnitrosamine, thioacetamine, and D-galactosamine. We have selected CCl4 because it has been reported to promote a cirrhosis that is remarkably similar to alcoholic cirrhosis in humans (2). However, numerous protocols that differ in intoxication times and/or administration routes have been described. The unpredictable response of animals to CCl4 makes it difficult to produce a consistent model of cirrhosis with this drug. Most often, CCl4 administration to rats must be maintained for up to 3 wk to produce a steatosis and for up to 10 wk more to obtain fibrosis or cirrhosis. After 10-12 wk of CCl4 treatment, liver regeneration after a resection has been described to be impaired and delayed (2, 18) as inferred from [3H]thymidine incorporation into DNA as a marker of cell proliferation. We then expected that this impaired regenerative process would be further reflected by an altered expression of cell cycle-associated genes, such as those for cyclins that are assumed to regulate cell cycle progression in the regenerating liver (16). Therefore, after 16 wk of CCl4-induced cirrhosis as confirmed by histological examination, we studied the expression patterns of the cyclins B, D1, and E, which are the essential components of the cell cycle machinery. There was a delayed and decreased expression of the mRNA for these cyclins, notably for cyclin E. Likewise, the TK mRNA levels that are strictly proportional to the levels of TK activity during liver regeneration (33) showed an impaired expression after PH in our cirrhotic animals, with a delayed peak at day 3 postresection. Together, these results provide evidence for the impaired and delayed hepatic regeneration in our model of CCl4-induced cirrhosis.
Before surgery, CCl4-induced
cirrhosis was associated with a significant increase in the
intrahepatic mRNA level for TGF-. This observation reflects the
chronic hepatic regeneration that constitutes an important component of
the cirrhotic process (25) and focuses on the role of this growth
factor in the pathogenesis of hepatic regeneration in cirrhosis.
After PH in healthy rats, changes in intrahepatic HGF, TGF-, and
c-Met mRNA levels showed a classical pattern, as already described by
several studies (8, 20). The peak of c-Met mRNA expression was earlier,
at 6 h, preceding that of HGF.
In the CCl4-treated rats after PH in NPC, no significant change of HGF mRNA level was observed compared with that observed in healthy controls. In contrast, the significant decrease of both mRNA and protein levels of the HGF receptor, i.e., c-Met, is correlated with the impaired regeneration of cirrhotic liver. Therefore, despite a normal expression of HGF, the delayed proliferative process might be related to an impaired c-Met expression.
Our results suggest that after hepatectomy on cirrhotic liver, local
downregulation of c-Met mRNA and protein is crucial to regenerate
because it decreases the activity of this receptor/ligand system with a
lower targeting HGF action toward hepatocytes. Our observation is in
complete agreement with a recent study that mentioned the lack of c-Met
expression in fulminant hepatic failure livers associated with a poor
response to exogenous HGF that normally promotes liver growth and
regeneration (17). Other data have also been described (7) in which, in
this model of fulminant hepatic failure, lack of regenerative process
in the residual liver tissue was associated with elevated HGF and
TGF- levels and delayed expression of c-Met mRNA. In another way,
overexpression of c-Met played an important role in tumor growth (34)
and may be one of the several attributes that contributes to invasive tumor progression. Furthermore, a correlation between overexpression of
c-Met in HCC and patient survival (26) has been noted, whereas there
was no correlation between HGF concentration and this survival.
As also reported in humans by d'Errico et al. (6), our study suggests that, regardless of the level of liver HGF, c-Met protooncogene seems the most active modulator of liver cell proliferation, and its induction might be a critical factor in determining the site specificity of the receptor/ligand system and might be a useful prognostic marker for evaluating liver regeneration.
In the CCl4-treated group after
PH, we observed an early increase in TGF- mRNA level that remained
high up to day 6. Given that
constitutive TGF-
expression in transgenic mice increases hepatocyte
proliferation and leads to the development of hepatocellular tumors
(35), the sustained high expression seen in cirrhotic rat after PH
could likewise lead to the appearance of dysplasia or neoplasia in
chronically regenerating liver. Our data support the hypothesis by
Tomiya and Kenji (32) that TGF-
level may provide useful information
for diagnosis of postcirrhosis HCC. Cytokines constitute a complex
network of molecules involved in the regulation of the inflammatory
response (22) and coordinate physiological and pathological processes
in the liver, such as growth and regeneration (8), fibrosis, and
cirrhosis (29).
In our study, a strong increase in the mRNA levels for IL-1, IL-6,
and TNF-
, with a peak at 12 h, was observed in the isolated liver
NPC after PH in control rats, which suggests that a local inflammatory
response takes place in the liver after this injurious surgical process.
Moreover, hepatocyte DNA synthesis during liver regeneration has been
found to be suppressed in IL-6 /
mice (5). It has also
been proposed (20) that IL-6 and TNF-
are the major cytokines involved in liver regeneration and allow the
G0/G1
and G1/S transitions in the cell
cycle. We did not find any modifications in the intrahepatic mRNA
levels for IL-1
, IL-6, or TNF-
before PH in cirrhotic livers vs.
controls. However, a previous study (11) has shown a significant increase in the plasma levels of cytokines, which was correlated with
disease progress in liver cirrhosis and could be an easy way to follow
liver inflammation and fibrosis. Kamimura and Tsukamoto (14) has
observed an expression of hepatic TNF-
mRNA that was three- to
fourfold higher in ethanol-treated rats compared with controls. A
similar observation was made by Napoli et al. (24) who found a
significant activation of cytokines at the mRNA level in end-stage
cirrhosis in humans. Our results may be explained by
1) our experimental model, which is
quite different (CCl4-induced cirrhosis), and 2) an analysis of
isolated NPC instead of the whole liver cells. Therefore, the
expression of cytokine mRNA in liver cirrhosis might depend on the
status of liver disease at the time of evaluation and the origin of the
secreted cytokines.
The IL-6 and TNF- mRNA levels were significantly decreased and
delayed in cirrhotic rats after PH. These significant intrahepatic cytokine decreases and the resulting delay in cell-cycle progression may account for the impaired regeneration observed in cirrhotic liver
after surgery. In contrast, IL-1
, described as a downregulator of
liver regeneration (1), did not show in cirrhotic livers a decrease of
its mRNA level but only a delayed peak at days
1-3 compared with results observed after PH on
normal livers. Thus the time course of IL-1
expression is difficult
to reconcile with the kinetics of regeneration.
In conclusion, our study suggests that, even though cytokines and growth factors play a role in the impaired regeneration of cirrhotic livers after PH, the low expression of the c-Met/HGF receptor along with a normal expression of its specific ligand may provide a further explanation for this delayed regenerative process. Our data now stress the role of the c-Met protooncogene as a critical partner of liver cell regeneration. This observation eventually could provide a basis for further investigations aimed at a therapeutic use of these factors.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Masson, INSERM U519, Faculté de Médecine et de Pharmacie, 22 Bd Gambetta, 76 183 Rouen, France.
Received 16 November 1998; accepted in final form 2 June 1999.
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