University of Groningen
Liver fibrosis in vitro
Bovenkamp, Marja van den
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2006
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Bovenkamp, M. V. D. (2006). Liver fibrosis in vitro: Liver slices as a promising alternative s.n.
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Chapter 4
Effect of pentoxifylline on stellate cell
activation in human liver slices
M. van de Bovenkamp
G.M.M. Groothuis
D.K.F. Meijer
P. Olinga
Chapter 4
Abstract
Introduction: Precision-cut liver slices are a promising alternative to cell culture models
for the study of hepatic stellate cell (HSC) activation and fibrogenesis since HSC are
maintained in their original extracellular matrix and multicellular milieu. In the present
study we evaluated human liver slices as a tool to study fibrogenesis and to test antifibrotic drugs.
Methods: Firstly we determined whether spontaneous fibrogenesis occurred in human
liver slices (250 µm thick, diameter 8 mm) during 1-48 hours of incubation and whether
the anti-fibrotic compound pentoxifylline could prevent this. Secondly, the effect of
pentoxifylline on carbon tetrachloride (CCl4)-induced early HSC activation was
determined. To assess HSC activation and fibrogenesis mRNA expression of several
markers was measured.
Results: After prolonged incubation of human liver slices (> 24 hours), αSMA and procollagen 1a1 mRNA expression started to increase, which was significantly inhibited by
pentoxifylline. Analysis of synaptophysin and fibulin-2 mRNA expression during
incubation with or without pentoxifylline indicated that likely both HSC and other
(myo)fibroblasts are involved in this fibrotic process. CCl4-induced early HSC
activation, which was reflected by increased HSP47 and αB-crystallin mRNA
expression, was not inhibited by pentoxifylline.
Conclusions: 1) Preparation and/or culturing of human liver slices induced fibrogenesis
after prolonged incubation, which may be mediated by both activated HSC and resident
liver (myo)fibroblasts. 2) Our results suggest that pentoxifylline can only inhibit
relatively late processes of HSC activation. 3) Human liver slices are a promising in vitro
system to study both early HSC-activation and fibrogenesis, and to test anti-fibrotic
drugs in a multicellular environment, closely resembling the in vivo situation in man.
58
Effects of pentoxifylline
Introduction
Liver fibrosis due to viral, metabolic, or alcohol-induced liver injury is one of the
leading causes of death worldwide [1]. To date no curative treatment for liver fibrosis is
available and patients are dependent on liver transplantations. In order to develop
effective anti-fibrotic pharmaco-therapies, it is of importance to elucidate the
mechanisms underlying fibrosis development in human liver. In addition, there is a
great need for representative experimental models to test the effects of potential antifibrotic compounds. In vivo animal models for liver fibrosis are time-consuming and
implicate high discomfort for the experimental animals. Therefore, the first stage in the
testing of a potential anti-fibrotic drug is to study its efficacy in in vitro models. As
hepatic stellate cell (HSC) activation is a key event in fibrosis development [1, 2],
current in vitro models for fibrosis mainly encompass cultures of primary HSC or HSC
cell lines, occasionally co-cultured with other liver cell types. These models have
contributed significantly to the understanding of HSC biology, but cannot accurately
incorporate in vivo cell-cell and cell-extracellular matrix interactions, which play an
important role in the multicellular fibrosis process. In addition, although activated HSC
are thought to play the major role in fibrosis development, the (myo)fibroblast
population involved in fibrosis development in vivo, is likely more heterogeneous, and
may also involve resident myofibroblasts, portal fibroblasts, second-layer cells, and bone
marrow derived myofibroblasts [3, 4]. Current cell culture models do not account for
this heterogeneous population of fibrogenic cells. Thus, there persists a need for an in
vitro model that can mimic the in vivo situation more closely. Recently, precision-cut
liver slices came into attention as a promising alternative to cell culture models for the
study of fibrosis [5-9]. The main advantage of liver slices is that all liver cell types are
present and the cells are maintained in their original extracellular matrix and
multicellular milieu.
The applicability of liver slices for the study of the processes underlying HSC activation
and fibrogenesis was suggested in previous studies, which showed that during
incubation of rat liver slices spontaneous HSC activation and fibrogenesis occurs [6, 8].
In addition, we showed that the known fibrogenic compound carbon tetrachloride
(CCl4) induced early HSC activation in liver slices [5, 9]. The aim of the present study
was to evaluate human liver slices as a tool to study fibrogenesis and to test the effects
of anti-fibrotic drugs. For this purpose, first we studied whether spontaneous
fibrogenesis occurs during the incubation of human liver slices and evaluated the
involvement of HSC and (myo)fibroblasts in this process. Secondly, we determined the
effects of pentoxifylline on mRNA expression of several markers of fibrogenesis and
stellate cell activation during incubation of human liver slices. Finally, we studied
whether the previously described CCl4-induced early HSC activation in human liver
slices [9], which provides a model to study the mechanisms underlying toxicity-induced
HSC activation, can be inhibited by pentoxifylline.
As anti-fibrotic model compound pentoxifylline was chosen. Pentoxifylline is a
phosphodiesterase inhibitor, which inhibits proliferation and collagen synthesis in
cultured HSC and myofibroblasts [10-14], and reduces HSC activation and proliferation
in experimental animal models for fibrosis [15, 16]. In addition, pentoxifylline has anti59
Chapter 4
cytokine effects, mainly as inhibitor of interleukin 1 and tumor necrosis factor α
expression and activity [17, 18]. To assess the effects of control incubation and
incubation in the presence of CCl4 and/or pentoxifylline on HSC activation and
fibrogenesis in human liver slices, heat shock protein 47 (HSP47) and αB-crystallin
mRNA expression was used as an early marker for HSC activation, and expression of α
smooth muscle actin (αSMA) and pro-collagen 1a1 mRNA as a late marker for HSC
activation and fibrogenesis. Besides, to discern HSC related phenomena from those in
(myo)fibroblasts, synaptophysin and fibulin-2 mRNA expression was studied.
Synaptophysin is reported to be exclusively expressed in HSC, both quiescent and
activated [19]. Recently, in rat liver, fibulin-2 was reported to be almost exclusively
present in (myo)fibroblasts and rarely in activated HSC [20]. Therefore, in the present
study we also measured fibulin-2 mRNA expression in human liver slices, assuming that
this marker mainly reflects (myo)fibroblasts rather than activated HSC.
Methods
Human liver tissue
Human liver tissue was obtained from multi-organ donors (Tx-livers) or from patients after partial
hepatectomy because of metastasis of colorectal carcinoma (PH-livers). Consent from the legal authorities and
from the families concerned was obtained for the use of Tx-livers for transplantation-related research. The
Tx-liver was perfused with cold University of Wisconsin organ storage solution (UW, DuPont Critical Care,
Waukegan, IL, USA) in situ before explantation, and stored in cold UW until the liver was reduced in order to
perform reduced-size or split liver transplantation. During reduction the liver was immersed in UW cooled
with ice slush. The liver tissue remaining after bipartition was stored in cold UW solution until the start of
the slicing procedure. In case of PH-livers, consent to use liver tissue for research purposes was obtained from
the patients concerned. The technique of partial hepatectomy was performed as described earlier [21] after
which a wedge from the resected liver lobe was cut at distance from the metastases. Immediately after
excision the biopsy wedge was perfused with cold UW, and transported to the laboratory were the slicing
procedure was started within 30 min. The medical ethical committee of the University of Groningen
approved the research protocols.
Preparation of liver slices
Precision-cut liver slices (8 mm diameter, 250 µm thickness) were prepared in ice-cold Krebs-Henseleit buffer
saturated with carbogen (95% O2 / 5% CO2) and containing 25 mM glucose (Merck, Darmstadt, Germany), 25
mM NaHCO3 (Merck) and 10 mM HEPES (ICN Biomedicals, Inc. Aurora, OH, USA) using the Krumdieck
tissue slicer [22, 23]. Slices were stored at 4°C in UW until the start of the experiments.
Incubation of liver slices
Slices were pre-incubated for 1 hr in 3.2 ml Williams Medium E with glutamax-I (Gibco, Paisly, Scotland)
supplemented with 25 mM D-glucose and 50 µg/ml gentamycin (Gibco) (WEGG) under carbogen (95% O2/5%
CO2) atmosphere at 37°C in 6-well culture plates (Greiner bio-one, Frickenhausen, Germany) while gently
shaken. Pre-incubation enables the slices to restore their ATP-levels [24]. After pre-incubation slices were
transferred to 25 ml Erlenmeyer flasks containing 5 ml carbogen-saturated WEGG, and incubated at 37°C for
16 hours, while gently shaken. At the start of the incubation period 0, 5, or 10 µl carbon tetrachloride (CCl4,
Fluka Chemie, Steinheim, Switzerland) was added in the 20 ml headspace of the flask to a paper attached to
the stopper. Simultaneously 0 or 1mM pentoxifylline was added to the culture medium. During incubation
CCl4 evaporates, and equilibrium is reached between the gas phase and the medium. Alternatively, to study
marker expression profile in the liver slices during control incubation, after preincubation slices were
transferred to 6-wells culture plates containing fresh carbogen-saturated WEGG and incubated for 3-48
hours. In case of 48 hours of incubation, after 24 hours of incubation, slices were again transferred to 6-well
culture plates containing fresh WEGG and incubated for another 24 hours.
60
Effects of pentoxifylline
Viability
Each slice was transferred to a sonication solution containing 70% ethanol and 2 mM EDTA, snap frozen in
liquid nitrogen, and stored at -80°C until ATP determination. ATP was determined in the supernatant that
was obtained after sonication the samples 15 s. and centrifuging the homogenate 2’ at 13000 rpm using the
ATP Bioluminescence assay kit CLSII (Roche diagnostics, Mannheim, Germany).
Real-time PCR
RNA was isolated from three snap-frozen slices using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA
was synthesized from 2 µg total RNA, using the Promega Reverse Transcription System (Promega, Madison,
WI, USA). 0.63 µl cDNA was used in real-time PCR reactions using Taqman reaction mixture (Applied
Biosystems, Warrington, UK), and the appropriate primers and probes (Assays-On-Demand, Applied
Biosystems, Foster City, CA, USA). The comparative threshold cycle (CT) method was used for relative
quantification. CT is inversely related to the abundance of mRNA transcripts in the initial sample. Mean CT of
duplicate measurements was used to calculate the difference in CT for target and reference GAPDH gene
(∆CT), which was compared to the corresponding ∆CT of the control (∆∆CT). Data are expressed as foldinduction or repression of the gene of interest according to the formula 2-(∆∆CT).
Collagen protein determination
5 µm cryostat sections were stained with saturated picric acid containing 0.1% Sirius-Red for collagen protein
staining (Chroma, Stuttgart, Germany) and 0.1% Fast-Green for non-collagen protein staining (SigmaAldrich). To quantify the collagen content the color was eluted from the sections with 0.1 N NaOH diluted
1:1 with methanol and measured at 540 nm (Sirius-Red) and 605 nm (Fast-Green). Calculations were
performed according to Lopez-De Leon [25]. Firstly, the absorbance of Sirius-Red was corrected for the
contribution of Fast Green (29.1%). Secondly, the absorbances were divided by their color equivalences (2.08
and 38.4) to obtain values representing the amount of collagen and non-collagenous protein that were present
in the sections. Finally, the percentage of collagen per total protein was calculated.
Statistics
Experiments were performed with at least 3 livers using slices in triplicate from each liver. Data were
compared using an unpaired two-tailed Student’s-t-test. A P-value < 0.05 was considered significant. Data are
presented as mean ± standard error of the mean (SEM).
Results
ATP (arbitrary units)
Control incubation
Human liver slices remained viable during incubation, as indicated by essentially
constant ATP levels during the first 24 hours of incubation and a small, but significant
decrease after 48 hours of incubation (ATP retention 73 ± 10%) (Figure 1).
1.5
1.0
*
0.5
0.0
0
pre
3
6
16 24
Incubation time (hours)
48
Figure 1. ATP content of human liver
slices directly after the slicing procedure
(0) after pre-incubation (pre) and after 348 hours of incubation. ATP content is
expressed relative to the ATP content in
liver slices at the start of the incubation
period. The average of at least 5
independent experiments ± SEM is
shown. *: P< 0.05 as compared to slices
after pre-incubation.
61
Chapter 4
7
6
5
4
3
2
1
0
HSP47
*
Fold induction
Fold induction
To study the fibrotic process in human liver slices during incubation, the mRNA
expression level of several markers was measured. Expression of HSP47 and αBcrystallin mRNA, two early markers for HSC activation, was significantly increased at
the start of the incubation period and tended to normalize after prolonged incubation.
In contrast, mRNA levels of the late markers for HSC activation and fibrogenesis,
αSMA and pro-collagen 1a1, decreased during the first hours of incubation, whereas
after prolonged incubation expression started to increase as compared to the expression
levels after 16 and 6 hours of incubation, respectively. Fibulin-2 mRNA expression,
which was used as a marker for resident (myo)fibroblasts, also decreased during the first
hours of incubation, however, expression did not increase after prolonged incubation up
to 48 hours. Synaptophysin mRNA expression, which is a marker for HSC, remained
relatively constant during incubation, but significantly decreased after 48 hours of
incubation (Figure 2). Collagen protein content of the liver slices was determined in
two independent experiments and increased on average 1.5-fold during incubation
(Figure 3).
7
6
5
4
3
2
1
0
*
*
*
*
5
4
3
2
1
0
*
*
*
fibulin-2
1.0
*
0.0
0 pre 3 6 16 24 48
Incubation time (hours)
Fold induction
Fold induction
*
0 pre 3 6 16 24 48
Incubation time (hours)
synaptophysin
1.5
0.5
*
*
pro-collagen 1a1
*
Fold induction
Fold induction
αSMA
0 pre 3 6 16 24 48
Incubation time (hours)
2.0
*
0 pre 3 6 16 24 48
Incubation time (hours)
0 pre 3 6 16 24 48
Incubation time (hours)
1.0
0.8
0.6
0.4
0.2
0.0
αB-crystallin
1.0
0.8
0.6
0.4
0.2
0.0
HSP47
*
αB-Crystallin
*
αSMA
*
*
*
0 pre 3 6 16 24 48
Incubation time (hours)
Figure 2. mRNA expression of HSP47, αB-crystallin, αSMA, pro-collagen 1a1,
synaptophysin, and fibulin-2 in human liver slices directly after the slicing procedure (0),
after pre-incubation (pre), and after 3-48 hours of incubation. Expression levels are
indicated relative to expression levels in liver slices directly after the slicing procedure. The
average of at least 3 independent experiments ± SEM is shown. *: P< 0.05 compared to liver
slices directly after slicing.
62
Effects of pentoxifylline
Fold induction
2.0
1.5
1.0
0.5
0.0
0
6
24
48
Figure 3. Collagen protein content
as percentage of total protein
content in human liver slices after
different incubation intervals. Data
are expressed relative to collagen
protein content in liver slices
directly after the slicing procedure
The average of 2 independent
experiments ± standard deviation is
shown
Incubation time (hours)
The effect of pentoxifylline on the spontaneous fibrotic process in the liver slices was
determined by measuring marker expression after 16 and 24 hours of incubation with or
without pentoxifylline (Figure 4). mRNA expression of HSP47, αB-crystallin, and
synaptophysin was not inhibited by pentoxifylline at these time-points. In contrast,
mRNA expression of pro-collagen 1a1 and αSMA was significantly inhibited by
pentoxifylline both after 16 and 24 hours of incubation. Fibulin-2 mRNA expression
was inhibited after 24 hours of incubation with pentoxifylline but not after 16 hours of
incubation (Figure 4). ATP content of the liver slices was not influenced by
pentoxifylline (not shown).
Incubation in the presence of CCl4
Incubation of human liver slices in the presence of CCl4 resulted in a dose-dependent
decrease of the ATP content of the liver slices (Figure 5A). In slices incubated in the
presence of 10 µl CCl4, addition of pentoxifylline during the incubation period seemed
to increase the ATP content slightly, whereas ATP content of slices incubated with 0 or
5 µl CCl4 was unaffected by pentoxifylline (Figure 5B).
Previously we showed that CCl4 induces early HSC activation in human liver slices [5].
Similarly, in the present study CCl4 induced an increase in mRNA expression of HSP47
and αB-Crystallin, two early markers for HSC activation. mRNA expression of the late
markers for HSC activation and fibrogenesis αSMA and pro-collagen 1a1, was not
induced by CCl4. Synaptophysin mRNA expression, a marker for HSC, was not changed
after incubation with CCl4, whereas mRNA expression of fibulin-2, which was used as a
marker for (myo)fibroblasts, was significantly decreased after 16 hours of incubation
with CCl4 (Figure 6).
To determine whether CCl4-induced early HSC activation in human liver slices can be
inhibited, the liver slices were incubated simultaneously with CCl4 and the anti-fibrotic
compound pentoxifylline, and effects on mRNA expression of the above-mentioned
markers was measured. Pentoxifylline did not inhibit the CCl4-induced increased
mRNA expression of αB-crystallin or HSP47 in human liver slices, whereas expression
of αSMA and pro-collagen 1a1 mRNA was significantly reduced. mRNA expression of
synaptophysin and fibulin-2 in liver slices incubated with CCl4 was not affected by
pentoxifylline (Figure 6). The effects of pentoxifylline on liver slices incubated without
CCl4 are indicated in figure 4.
63
Chapter 4
24 hours of incubation
16 hours of incubation
2.0
Fold induction
Fold induction
2.0
1.5
1.0
0.5
1.0
0.5
0.0
0.0
0
1
PTX (mM)
0
1
PTX (mM)
4.0
Fold induction
Fold induction
4.0
3.0
2.0
1.0
2.0
1.0
0
1
PTX (mM)
0
1
PTX (mM)
1.0
*
0.6
0.4
0.2
Fold induction
Fold induction
1.0
0.8
0.6
0.4
0
1
PTX (mM)
1.0
0.8
*
0.4
0.2
Fold induction
Fold induction
1.0
0.8
pro-collagen 1a1
0.6
0.4
0.2
*
0.0
0.0
0
1
PTX (mM)
0
1
PTX (mM)
2.5
Fold induction
2.5
Fold induction
*
0.2
0
1
PTX (mM)
2.0
1.5
1.0
0.5
synaptophysin
2.0
1.5
1.0
0.5
0.0
0.0
0
1
PTX (mM)
0
1
PTX (mM)
1.5
Fold induction
1.5
Fold induction
αSMA
0.0
0.0
0.6
αB-Crystallin
3.0
0.0
0.0
0.8
HSP47
1.5
1.0
0.5
fibulin-2
1.0
0.5
*
0.0
0.0
0
1
PTX (mM)
0
1
PTX (mM)
Figure 4. mRNA expression of HSP47, αB-crystallin, αSMA, pro-collagen 1a1,
synaptophysin, and fibulin-2 in human liver slices incubated for 16 or 24 hours in the
presence or absence of 1 mM pentoxifylline (PTX). Expression levels are indicated relative
to expression levels in liver slices incubated without pentoxifylline. The average of at least 5
independent experiments ± SEM is shown. *: P< 0.05 compared to slices incubated without
pentoxifylline.
64
Effects of pentoxifylline
A
B
250
ATP (% of control)
ATP (% of control)
120
100
80
60
*
40
20
0
PTX
5 µl
CCl4
control
200
150
100
50
0
10 µl
CCl4
1 PTX (mM) 0
,0
5 µl CCl4
1
10 µl CCl4
Figure 5. A) ATP content of human liver slices after 16 hours of incubation in the presence
of pentoxifylline (PTX, 1 mM) or CCl4 relative to the ATP content in liver slices after 16
hours of control incubation B) ATP content of human liver slices after 16 hours of
incubation with 5 or 10 µl of CCl4 in the presence of 1 mM pentoxifylline (PTX) relative to
ATP levels in liver slices incubated with CCl4 alone. The average of at least 5 independent
experiments ± SEM is shown. *: P< 0.05 compared to control slices.
3
3
2
*
1
0
αB-Crystallin
2
*
1
CCl4
+
CCl4
0.0
CCl4
CCl4
+
PTX
+
fibulin-2
*
2.5
Fold induction
0.5
CCl4
PTX
synaptophysin
Fold induction
1.0
CCl4
+
2.0
*
control
control
CCl4
PTX
pro-collagen 1a1
1.5
*
0.0
control
CCl4
*
0.5
0
control
αSMA
1.0
PTX
Fold induction
1.5
Fold induction
HSP47
Fold induction
Fold induction
4
1.5
1.0
0.5
0.0
2.0
1.5
1.0
0.5
0.0
control
CCl4
CCl4
+
PTX
control
CCl4
CCl4
+
PTX
Figure 6. mRNA expression of HSP47, αB-crystallin, αSMA, pro-collagen 1a1,
synaptophysin, and fibulin-2 in human liver slices incubated for 16 hours in the absence
(control) or presence (CCl4) of 5 µl CCl4, and in liver slices incubated simultaneously with
5µl CCl4 and 1 mM pentoxifylline (CCl4+PTX). Expression levels are expressed relative to
expression levels in liver slices incubated with CCl4. The average of 6 independent
experiments ± SEM is shown. *: P< 0.05.
65
Chapter 4
PDGF receptor mRNA expression in human liver slices
Because the anti-fibrotic effects of pentoxifylline are generally attributed to its effects
on platelet derived growth factor (PDGF) signaling, mRNA expression levels of the
receptor for PDGF in human liver slices was determined as an indication of the
responsiveness to PDGF. During control incubation of human liver slices PDGF
receptor mRNA levels initially decreased and started to increase after prolonged
incubation (Figure 7A). Incubation in the presence of CCl4 for 16 hours did not
influence PDGF receptor mRNA expression in human liver slices (Figure 7B).
A
B
4
1.5
Fold induction
Fold induction
#
3
2
1
*
0
1.0
0.5
0.0
0
pre
3
6
16 24
Incubation time (hours)
48
control CCl4
Incubation
Figure 7. A) mRNA expression of the PDGF receptor in human liver slices directly after the
slicing procedure (0), after preincubation (pre), and after 3-48 hours of incubation.
Expression levels are indicated relative to expression levels in liver slices directly after the
slicing procedure (0). B) mRNA expression of the PDGF receptor in human liver slices
incubated for 16 hours in the presence of 5 µl CCl4 relative to expression levels in slices
incubated without CCl4 (control). The average of at least 3 independent experiments ± SEM
is shown. *: p<0.05 compared to liver slices directly after slicing #: P<0.05 compared to liver
slices after 3 hours of incubation.
Discussion
Recently the applicability of liver slices for the study of the processes underlying HSC
activation and fibrogenesis was suggested in several studies [5-9]. The main advantage
of liver slices over current available in vitro models for fibrosis is that all liver cell types
are present in a physiologic extracellular matrix. In the present study we aimed to
evaluate human liver slices as a tool to study fibrogenesis and as a test system for antifibrotic drugs. For this purpose we first studied spontaneous fibrogenesis in the liver
slices and the involvement of HSC and/or resident (myo)fibroblasts, and determined the
effects of pentoxifylline on this process. Secondly, we studied whether CCl4-induced
early HSC activation in human liver slices could be inhibited by pentoxifylline.
HSC activation and fibrogenesis in human liver slices during control incubation
During incubation of human liver slices an initial decrease in αSMA and pro-collagen
1a1 mRNA expression was observed in the liver slices, which was also described
previously in rat liver slices [6, 8], but is yet unexplained. In the present study this
66
Effects of pentoxifylline
decreased expression was also observed for fibulin-2 mRNA expression. In rat liver this
marker is mainly present in (myo)fibroblasts [20], and we assumed that also in human
liver this marker mainly reflects (myo)fibroblasts rather than activated HSC. In contrast
to fibulin-2 mRNA expression, synaptophysin mRNA expression, which is specific for
HSC [19], did not show an initial decrease during incubation. These results suggests that
(myo)fibroblasts are at least partly responsible for the decrease of αSMA and procollagen 1a1 mRNA expression during incubation and that the decreasing expression of
αSMA is not due to loss of HSC, which are known to express αSMA in normal human
liver [26]. However, a specific effect on αSMA expression in HSC cannot be excluded.
In contrast to the late markers for HSC activation and fibrogenesis, mRNA expression of
the early markers HSP47 and αB-crystallin was increased in human liver slices during
the first hours of incubation. During the incubation period the mRNA expression of αBcrystallin remained approximately 2-fold increased, although non significantly, whereas
HSP47 mRNA expression normalized after prolonged incubation. The latter may be
related to the observation that in progressed fibrosis in patients HSP47 protein, rather
than mRNA expression is increased in HSC [27]. After prolonged incubation of human
liver slices also the mRNA expression of αSMA and pro-collagen 1a1 as well as the
collagen protein content in human liver slices started to increase. Similar changes in
expression of fibrogenic markers in liver slices were also described previously in rat
liver slices [6, 8] and suggest that spontaneous activation of HSC and/or fibroblasts and
fibrogenesis occurs in the liver slices. Alternatively, it may reflect an increased number
of (myo)fibroblasts or (activated) stellate cells in the liver slices. Because fibulin-2 and
synaptophysin mRNA expression did not increase during incubation this seems less
probable, however, the inhibitory effect of pentoxifylline on fibulin-2 expression after
24 hours of incubation could indicate some involvement of (myo)fibroblasts in the
spontaneous fibrotic process in human liver slices.
Pentoxifylline did not inhibit the early HSC activation reflected by the initial increased
expression of αB-crystallin and HSP47 mRNA in human liver slices, but significantly
inhibited the increase in mRNA expression of αSMA and pro-collagen 1a1 in the liver
slices after prolonged incubation. These results suggest that pentoxifylline can only
inhibit relatively late stages of HSC activation, which may be related to the mechanism
of action of the compound. The anti-fibrotic effects of pentoxifylline are generally
attributed to inhibition of platelet derived growth factor (PDGF) signaling via the
elevation of intracellular cyclic adenosine mono-phosphate levels due to inhibition of
phosphodiesterases [17]. PDGF is the most potent inducer of HSC and (myo)fibroblast
proliferation in culture and plays a role in the perpetuation of HSC activation [2]. Since
only activated HSC express the PDGF receptor, responsiveness of HSC to PDGF may
require that the cells are fully activated [28]. If the inhibitory effect of pentoxifylline is
solely mediated via its effect on PDGF signaling, this could explain why it is unable to
inhibit early stages of HSC activation. Indeed, PDGF receptor mRNA expression in
human liver slices paralleled the mRNA expression of the late markers for fibrosis
αSMA and pro-collagen 1a1, rather than that of the early markers HSP47 and αBcrystallin. However, it was also reported that pentoxifylline inhibits HSC activation
67
Chapter 4
independent of its phosphodiesterases inhibitory activity [15], thus its effect may not
exclusively be mediated via interfering with PDGF signaling.
The inhibitory effect of pentoxifylline on αSMA and pro-collagen 1a1 mRNA
expression in human liver slices was observed at a time-point at which mRNA
expression was not yet increased compared to expression levels in slices prior to
incubation. This suggests that the fibrogenic process in human liver slices occurs
already early during incubation, but is masked by the simultaneously occurring process
that leads to decreased expression of αSMA and pro-collagen 1a1 mRNA. This
hypothesis is supported by the early increase of HSP47 and αB-crystallin mRNA and
the slightly increased collagen protein content of the liver slices after 6 hours of
incubation. The underlying mechanism of these processes remains to be elucidated but
is likely intrinsic to the preparation and culturing of the slices. Factors in the method
used that might trigger the fibrotic process are ischemia-reperfusion injury, the
presence of high oxygen tension during incubation, cell death during incubation, or
accumulation of (waste) products in the slices. Pilot experiments suggested that the
damage to the cells on the surface caused by the cutting of the slices does not play a
major role in triggering the spontaneous fibrotic process in liver slices and that
accumulation of bile salts, which is potentially fibrogenic, did not occur during
incubation of the liver slices. Additional evaluation of the liver slices is necessary to
further elucidate this matter.
CCl4-induced early HSC activation in human liver slices
Previously, we showed that early HSC activation could be induced in human liver slices
via a multicellular mechanism by incubation with the fibrogenic compound CCl4 [9]. To
evaluate this model as a system to test the effects of anti-fibrotic compounds, in the
present study the liver slices were incubated simultaneously with CCl4 and the antifibrotic compound pentoxifylline. Firstly, the effect of pentoxifylline on the CCl4induced decrease of ATP content was determined, showing a slightly, though not
significantly, improved ATP content when liver slices were incubated with 10 µl CCl4
in the presence of pentoxifylline. In vivo liver toxicity of CCl4 is caused by cytochrome
P450 mediated conversion of CCl4 into free radicals in the hepatocytes [29].
Pentoxifylline has anti-oxidant properties [30], which could explain this small
protective effect of pentoxifylline on CCl4-induced toxicity. To avoid that the possible
effect on CCl4-toxicity interferes with the measurement of potential inhibitory effects
of pentoxifylline on CCl4-induced early HSC activation, we choose to study the latter in
liver slices incubated with 5 µl CCl4. At this concentration no beneficial effect of
pentoxifylline on CCl4-toxicity in human liver slices was observed.
As reported previously [9], incubation of human liver slices with the fibrogenic
compound CCl4 resulted in an increase in mRNA expression of HSP47 and αBcrystallin, two early markers for HSC activation [27, 31]. The mRNA expression of
αSMA and pro-collagen 1a1, which is induced later in the process of HSC activation
[32, 33], was not changed after 16 hours of incubation with CCl4 as compared to control
incubation. Synaptophysin mRNA expression, which is a marker for quiescent as well as
activated HSC, was also not affected by CCl4. These results indicate that the increased
expression of the early HSC activation markers is not caused by an increased number of
68
Effects of pentoxifylline
HSC or (myo)fibroblasts present in the liver slices and that early HSC activation was
induced. The reduced expression of fibulin-2 after incubation with CCl4 may be
explained by cell death. In rat liver, fibulin-2 is expressed in (myo)fibroblasts in the
portal area as well as in cells surrounding the central vein [20], the latter is the primary
localization of CCl4 toxicity [29].
Pentoxifylline did not inhibit the CCl4-induced increased expression of the early
markers for HSC activation HSP47 and αB-crystallin, nor did it inhibit synaptophysin
or fibulin-2 mRNA expression after 16 hours of incubation. In contrast, mRNA
expression of αSMA and pro-collagen 1a1 was significantly inhibited by pentoxifylline,
despite the absence of CCl4-induced increased mRNA expression of these markers.
These results show that pentoxifylline is unable to inhibit early HSC activation in
human liver slices, and suggest that pentoxifylline does have inhibitory effects on fully
activated HSC and/or other (myo)fibroblast populations in human liver slices. This
corresponds with the effects of pentoxifylline on the spontaneously occurring fibrotic
process in human liver slices. As discussed above, the inhibitory effects of pentoxifylline
on HSC may require that the cells express the PDGF receptor. In human liver slices
PDGF receptor mRNA expression was not increased after 16 hours of incubation with
CCl4, which could indicate that the PDGF receptor is not yet expressed. If the
inhibitory effect of pentoxifylline is solely mediated via its effect on PDGF signaling,
this could explain why pentoxifylline is unable to inhibit the CCl4-induced early stages
of HSC activation.
In conclusion
Our results show that preparation and/or culturing of human liver slices induced
fibrogenesis after prolonged incubation, which may be mediated by both activated HSC
and resident liver (myo)fibroblasts. Pentoxifylline inhibited this spontaneous fibrogenic
process in human liver slices but did not inhibit CCl4-induced early activation of HSC.
These results suggest that pentoxifylline can only inhibit relatively late processes of
HSC activation. Therefore, to confirm that CCl4-induced early HSC activation can also
be used as a model to study effects of anti-fibrotic drugs, other types of inhibitors,
which act on different pathways involved in HSC activation, should be tested. Taken
together, human liver slices provide a promising in vitro system to study both early
HSC activation and fibrogenesis and to test anti-fibrotic drugs in a multicellular
environment, closely resembling the in vivo situation in man.
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