Department of Internal Medicine, Section of Gastroenterology and Endocrinology, University of Göttingen, 37075 Göttingen, Germany
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ABSTRACT |
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Hepatic
stellate cells (HSC), particularly activated HSC, are thought to
be the principle matrix-producing cell of the diseased liver. However,
other cell types of the fibroblast lineage, especially the rat liver
myofibroblasts (rMF), also have fibrogenic potential. A major
difference between the two cell types is the different life span under
culture conditions. Although nearly no spontaneous apoptosis
could be shown in rMF cultures, 18 ± 2% of the activated HSC
(day 7) were apoptotic. Compared with activated
HSC, CD95R was expressed in 70% higher amounts in rMF. CD95L could
only be detected in activated HSC. Stimulation of the CD95 system by
agonistic antibodies (1 ng/ml) led to apoptosis of all rMF
within 2 h, whereas activated HSC were more resistant (5.3 h/ 40%
of total cells). Although transforming growth factor- downregulated
apoptosis in both activated HSC and rMF, tumor necrosis
factor-
(TNF-
) upregulated apoptosis in rMF. Lack of
spontaneous apoptosis and CD95L expression in rMF and the
different reaction on TNF-
stimulation reveal that activated HSC and
rMF belong to different cell populations.
CD95 receptor; CD95 ligand; transforming growth factor-
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INTRODUCTION |
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ACTIVATION OF HEPATIC
STELLATE cells [HSC; so called since the consensus conference of
1996 (1)], also known as Ito cells, lipocytes, vitamin
A-storing cells, perisinusoidal cells, and liver pericytes, is supposed
to be one of the most important steps in liver tissue repair and in
development of liver fibrosis. Activation describes the morphologic
transformation of a vitamin A-storing quiescent cell through an
intermediate state (transitional HSC) to a myofibroblast-like activated
HSC. Activated HSC are thought to represent the major matrix-producing
cell during repair after acute damage and liver fibrogenesis in case of
chronic damage. However, activated HSC show characteristics commonly
known for smooth muscle cells and myofibroblasts. In vitro and in
vivo they have been shown to produce the intermediate filaments
vimentin, desmin, and the myofilament smooth muscle -actin (SMA)
(10, 13, 33, 38, 39, 41, 46, 52).
Conversely, other cell types of the fibroblast lineage (ie., interstitial fibroblasts, vascular myofibroblasts, and bile duct epithelial cells) have also been shown to be of particular fibrogenic importance, especially in early stages of cholestatic and serum-induced hepatic fibrosis models (4, 12, 14, 18, 47-49, 53). Although a method for isolation of rat liver myofibroblasts (rMF) has already been described in 1985 by Leo et al. (30) and a few other in vitro studies (3, 17, 40) exist demonstrating that activated HSC and rMF may be regarded as cell populations of different origin, both having fibrogenic potential, the possible involvement of the rMF in the fibrosis process has often been disregarded, mainly because rMF has been thought to be the result of a transdifferentiation of the HSC. This may be due to the lack of specific markers to differentiate activated HSC from other fibroblastic cells in vivo. Because activated HSC express SMA (39), SMA-positive cells detected in damaged rat and human livers are thought to be activated HSC. Furthermore, SMA gene expression is considered an identification marker for HSC in vitro. Knittel et al. (25) recently strongly suggested that rat HSC cultures may contain few fibulin-2-/SMA-positive cells whose increase in number during time leads to the overgrowth of nonproliferating HSC. Because this could be the explanation for the supposed transdifferentiation, establishment and characterization of rMF primary culture were necessary.
HSC are known to undergo CD95-mediated spontaneous apoptosis
when they are activated (42). To study the mechanisms
underlying the survival advantage of myofibroblasts in vitro, we
analyzed primary cultured HSC as well as primary and long-term cultured rMF with respect to apoptosis behavior. Our data show that,
whereas rMF can be passaged several times, HSC developed spontaneous
apoptosis within the activation period. This may be due to the
phenomenon that rMF possess CD95R but lack CD95L, which, in contrast,
is expressed by activated HSC. Further results also show different apoptosis behavior of rMF and activated HSC after treatment
with the CD95 agonistic antibody and tumor necrosis factor (TNF-) stimulation. The higher CD95R expression and the lack of CD95L expression of the rMF cultures and their behavior on cytokine stimulation may be crucial for the survival of these cells in vivo
during chronic liver damage. Further studies will allow understanding of the interaction between apoptotic mechanisms and cell cycle control points. It will also be interesting to study the TNF-
pathway of apoptosis induction in myofibroblasts compared with its antiapoptotic effect in activated HSC.
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MATERIALS AND METHODS |
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HSC and rMF Isolation: Characterization, Plating, and Culture Conditions
Wistar rats were provided by Charles River (Sulzfeld, Germany) and maintained under 12:12-h light/dark cycles with food and water ad libitum. During research described in this report, all animals received humane care in accordance with institutional and National Institutes of Health guidelines.HSC were isolated by sequential in situ perfusion with collagenase and pronase, as previously described (19-23, 26, 27). HSC (40 × 106) were obtained as mean per rat.
Cells were plated onto 24-well Falcon plates (Becton Dickinson, Heidelberg, Germany), 35-mm petri dishes (Greiner, Krefeld, Germany), 96-well Falcon plates (Becton Dickinson) or Lab Tek tissue culture slides (Nunc, Naperville, IL) with a density of 30,000 cells/cm2. Cells were cultured in DMEM supplemented with 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. Culture medium was replaced 2 days after plating and then every other day. Cells were kept in culture at 37°C in 5% CO2 atmosphere and 100% humidity.
To evaluate purity of cultures, HSC were tested by immunofluorescence
at day 0, day 2 (quiescent HSC/early activated
HSC), and day 7 (activated HSC) after plating as described
previously (19-23, 26, 27). Contamination with
Kupffer cells (ED1 positive) was <2%, and neither endothelial
cells nor hepatocytes were detected. With the use of SMA
immunoreactivity as an activation parameter, HSC were fully activated
after 7 days of primary culture (100% SMA positive). Fibulin-2
positive cells were always <1%. Some of the cultures were kept up to
2 or 3 wk and were either fixed and stored at 20°C until staining
was performed or trypsinized and passaged several times.
For isolation of rMF, the liver was enzymatically digested as described above. The nonparenchymal liver cell population was separated by a Nycodenz density gradient, and the fraction consisting of Kupffer cells and sinusoidal endothelial cells was further purified by centrifugal elutriation according to Knook et al. (28) and De Leeuw et al. (8, 9). With the use of a JE-6B elutriation rotor in a J2-21 centrifuge (Beckman Instruments, Palo Altro, CA) at 2,500 rpm, a fraction enriched with rMF was collected at a flow rate of 23 ml/min. rMF (60 × 106) were obtained as mean per rat. HSC containing the typical fat droplets could not be detected by light microscopy. The presence of so-called empty HSC not containing typical vacuoles cannot totally be excluded, however, because empty HSC at least should be positive for one of the marker proteins like desmin, glial fibrillary acidic protein (GFAP), or V-CAM-1 (35), which were undetectable (GFAP) or present in <1.5% (desmin) or 0.5% (V-CAM-1) of the isolated cells. A considerable contamination of the 23-ml/min fraction with empty HSC is unlikely.
Cells of the 23-ml/min fraction were plated onto 24-well Falcon plates (Becton Dickinson) at a density of 30,000 cells/cm2. Cells were cultured in DMEM supplemented with 15% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1% L-glutamine. At confluency, usually reached within 7-10 days, cells were released from the culture plates by trypsination and were replated at a 1:4 split ratio. rMF were passaged again at confluency using the same experimental condition. Subcultivation was performed several times.
Flow Cytometric and Fluorescence Microscopic Quantification of Living, Apoptotic and Necrotic HSC or rMF
For quantification of apoptotic cells, we used flow cytometry after trypsination of HSC and rMF (Epics ML; Coulter, Kerfeld, Germany). To detect apoptotic changes, staining with annexin V-FITC/propidiumiodide and Tdt-mediated X-dUTP nick-end labeling (TUNEL) were used (Boehringer Mannheim, Indianapolis, IN). Data obtained by TUNEL labeling were identical to those obtained with the annexin V-FITC/propidium iodide binding.Western Blot Analysis of SMA, Fibulin-2, CD95, CD95L, and TNF Receptors 1 and 2
Cells at different times after plating were lysed in hot Laemmli buffer (95°C) and processed by SDS-PAGE under reducing conditions according to Laemmli (29). Protein content of cellular lysates was calculated by the Coomassie protein assay (Pierce, Rockford, IL). Proteins were transferred onto Hybond-enhanced chemiluminescense nitrocellulose hybridization transfer membranes according to Towbin et al. (51). Immunodetection was performed according to the enhanced chemiluminescense Western blotting protocol. Antibodies against SMA (Sigma, Munich, Germany), fibulin-2 (Dr. R. Timpl, MPI for Biochemistry, Martinsried, Germany), CD95 (APO-1/Fas), CD95L, TNF receptor (TNFR)1, and TNFR2 (Santa Cruz Biotechnology, Santa Cruz, CA) were used at 2.5 µg/ml solutions, and peroxidase-labeled anti-mouse and anti-rabbit immunoglobulins (DAKO, Copenhagen, Denmark) were each used at a 1:1,000 dilution. Densitometric evaluation of the blots was performed using the program Scion Image version beta 2 (National Institutes of Health).Northern Blot Analysis of SMA and Fibulin-2
Briefly, 5 µg of total RNA was resolved by agarose gel electrophoresis, transferred to nylon membranes, and hybridized with specific 32P-labeled cDNA probes. Hybridization was performed for 2 h at 68°C using the QuickHyb Kit (Stratagene, La Jolla, CA). Posthybridization washes were performed two times for 15 min each at 60°C in 2× standard saline citrate solution containing 0.1% SDS. Nylon filters were washed, dried, and exposed to X-ray films atImmunocytochemical Detection of SMA, Fibulin-2, CD95R, and CD95L
First transmission pictures of HSC (days 2 and 7) and rMF (passage 4) were taken after marking the regions of interest. After cell cultures were fixed in methanol/acetone (5 min/10 s atInvestigation of Soluble CD95 and CD95L.
To investigate whether rMF releases soluble CD95R and CD95L, we performed Western blot analysis. For this purpose, we used concentrated supernatants (30-min vacuum centrifugation) of rMFs that were handled as described above. To functionally prove the possible presence of CD95L in the supernatant, we cultivated HSC in the quiescent phase (day 2) in the presence of supernatants of rMF. Occurrence of apoptosis was then measured by flow cytometry. As a second method to detect soluble CD95L, we performed a sCD95L ELISA (Boehringer Mannheim) according to the manufacturer's protocol using supernatants from rMF and HSC cultures.Occurrence of Spontaneous and Induced Apoptosis in HSC and rMF
To investigate induction of apoptosis and apoptosis occurring spontaneously in activated HSC (day 7) and rMF, we performed a test using confocal laser scan microscopy (Zeiss) in the time-scan mode. To detect early apoptotic changes, staining with annexin V-FITC was used. To distinguish apoptosis and necrosis, annexin V-FITC and trypan blue, a common dye exclusion test, were employed in parallel for showing membrane integrity after annexin V-FITC binding to cells. In all investigated cases, we could not notice loss of membrane integrity within 30 min after annexin V-FITC binding was detected; but 12 h after initiation of the test, considerable amounts of cells showed membrane leakage indicating secondary necrosis.Activated HSC and rMF were treated with 3 and 1 ng/ml of CD95 agonistic antibodies, respectively (Bender Med Systems, Ingelheim, Germany). Preliminary dose-response studies performed using different concentrations of CD95 agonistic antibodies (0.1, 0.5, 1, and 3 ng/ml) showed that triggering into apoptosis was obtained in activated HSC by 3 ng/ml and in rMF by 1 ng/ml. As negative controls, HSC and rMF were cultured with and without mouse IgG (Sigma). Apoptosis rates did not differ in the two controls.
Transfection Assay
rMF (primary culture, passages 2, 4, and 6) and activated HSC (day 7) were transfected by modification of the liposome transfection protocol. Briefly, rMF and HSC (day 7) were transfected with FuGENE (Boehringer-Mannheim) according to the manufacturer's protocol with minor modifications by using 10 µg CD95L plasmid DNA (PH Krammer, DKFZ, Heidelberg, Germany) per 2 × 106 cells. Null-plasmid controls were performed to evaluate a possible nonspecific effect of transfection assays on apoptosis of rMF or HSC. All experiments were repeated at least three times, and consistent results were obtained in all cases.Culture Conditions for Stimulation with Transforming Growth
Factor- or TNF-
Statistical Analysis
Results are expressed as means ± SD, and the significance of the difference between the means was assessed by the Mann-Whitney U-test. ![]() |
RESULTS |
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Characterization of rMF and HSC Isolations
Morphologically freshly isolated rMF do not show lipid droplets around the nuclei present in cytospins from freshly isolated HSC. Whereas freshly isolated rMF were >95% positive for SMA and fibulin-2, in HSC isolations, only a few SMA- and fibulin-2-positive cells could be detected (Fig. 1A). After activation (day 7), HSC express SMA but remain fibulin-2 negative (Fig. 1B). In HSC cultures derived from isolations with low yield of cells (5-7 Mio cells per rat liver marked with an asterisk; Fig. 1C), a contamination with fibulin-2-positive cells at day 2 (also SMA positive) and day 7 of culture could be detected (Fig. 1C). However, even in very pure HSC cultures (average yield of cells: 40-60 Mio cells per rat liver), single fibroblasts could be observed (Fig. 1D).
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Apoptosis of Activated HSC and rMF. Influence of the CD95 System
Different expression of CD95 and CD95L of activated HSC and rMF.
Initial experiments showed that rMF could be subcultivated without any
problems, whereas fully activated HSC could only be cultured until
passage 2. Regarding apoptosis 18 ± 2% of
activated HSC (day 7) could be shown to undergo spontaneous
apoptosis. In subcultivated HSC cultures 73.4% (passage
1) and 94.3% (passage 2) of evaluable cells showed
signs of apoptosis. In contrast to this, rMF cultures showed a
maximum of 1.3% apoptotic cells in all investigated cultures
(primary culture day 7; passages 2, 4,
and 6). Under the influence of 1 ng/ml CD95 agonistic
antibodies rMF cultures showed 100% evaluable cells undergoing
apoptosis, whereas in HSC cultures (day 7), 34.7%
showed apoptosis signs. A concentration of 3 ng/ml of CD95
agonistic antibodies was needed to completely trigger HSC cultures
(day 7) into apoptosis (Fig. 2).
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Transfection of rMF (passage 4) with CD95L.
To further investigate the role of CD95L in the apoptotic process
of rMF, cultures were transfected with a CD95L expressing vector.
Whereas control cultures and cultures transfected with the control
vector did not show a considerable induction of apoptosis, cultures transfected with the CD95L-cDNA underwent apoptosis
(95-100%) within 2 h. This apoptosis could be
avoided by treating the cells with CD95-blocking antibodies. To further
prove that apoptosis was due to the synthesized CD95L,
incubation of rMF with the supernatants of the CD95L-transfected rMF
cultures was performed. The supernatant also led to complete
apoptosis of untransfected rMF cultures. Simultaneous
administration of CD95-blocking antibodies completely prevented these
effects (Fig. 5). Supernatants from
CD95L-transfected rMF also induced apoptosis in cultures of
activated HSC (data not shown). Also, in this case, apoptosis
was prevented by the addition of CD95 antagonistic antibodies.
Furthermore, the presence of soluble CD95L in supernatants from
CD95L-transfected rMF was measurable by ELISA and Western blot. This
series of experiments was performed to show that transfection of rMF
with the CD95L plasmid truly induced synthesis of soluble and
biologically active CD95L.
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Time dependency of apoptosis induction due to CD95
agonistic antibodies.
To investigate the susceptibility of activated HSC and rMF to
CD95-mediated apoptosis induction, we used confocal laser scan microscopy in the time-scan mode. By these means, it is possible to
detect signs of early apoptosis over a 12-h period by taking one picture every quarter of an hour of the identical field. With the
use of CD95-agonistic antibodies (1 ng/ml), rMF cultures [primary culture (day 7), passages 2, 4, and
6] could completely be triggered into apoptosis
within 2 h, whereas activated HSC-cultures were more resistant.
Although, as shown above, 3 ng/ml of the CD95 agonist is necessary to
induce apoptosis of the total culture. The period of time until
activated HSC underwent the apoptosis process also was
considerably longer (5.5 h; Fig. 6).
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Effects of TNF- or TGF-
on Apoptosis of Activated HSC
and rMF.
Both TNF-
and TGF-
are fibrogenic mediators that have been shown
to exert the function of surviving factors in isolated HSC. In this
paper we confirmed previous results obtained on HSC (2, 23, 27,
37). With regard to apoptosis, TNF-
exerted effects
on rMF that differ from those observed on activated HSC. Whereas both
cytokines effected apoptosis inhibition on the latter cell type
(44), rMF showed a different but continuous behavior in
all investigated cultures. TNF-
administration led to a 10- to
20-fold increase in apoptosis in rMF. On the other hand,
TGF-
treatment caused an apoptosis inhibition in all cases.
After induction of CD95-mediated apoptosis by 1 ng/ml CD95
(rMF) or 3 ng/ml (HSC) agonistic antibodies (leading to 100%
apoptosis of all rMF or HSC cultures),
15% of total rMF
cultures could be rescued from apoptosis by TGF-
(Fig.
7).
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TNF-receptor status of activated HSC and rMF.
The pleiotropic biological properties of TNF- are signaled through
two distinct cell surface receptors [TNF receptor 1 (TNFR1) and
TNFR2]. To investigate whether a different TNFR status of rMF and HSC
is responsible for the different effect of TNF-
on apoptosis, we performed Western blot analysis. It could be
shown that the status of TNFR1 and TNFR2 was similar in activated HSC and rMF (primary culture, passages 2,
4, and 6). Moreover, it could be shown that the amount of
both receptors is not changed after TNF-
stimulation (Fig.
8).
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Effects of TGF- or TNF-
on CD95/CD95L expression of rMF.
To evaluate whether changes of the CD95/CD95L system are involved in
the effects of TGF-
or TNF-
, we performed Western blot analysis
of total proteins. Neither TGF-
nor TNF-
changed expression of
CD95 or CD95L (Fig. 9), suggesting that
TGF-
and TNF-
regulate apoptosis pathways in rMF
downstream of the CD95R and probably independently of the CD95 pathway
(caspase 3; data not shown).
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DISCUSSION |
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Until now, most studies in the field of liver repair and
fibrogenesis concentrated on activated HSC, which was regarded as the
major matrix synthesizing cell type. Only a few studies (3, 4, 7,
11, 12, 18, 24, 47, 49, 53) demonstrate that other
myofibroblastic cells also have fibrogenic potential. So far,
differentiation of activated HSC and other liver myofibroblasts hardly
has been possible because of morphologic similarities and lack of
specific marker proteins. Knittel et al. (25) recently demonstrated differences with respect to expression of cytoskeletal proteins, adhesion molecules, cytokine synthesis, and matrix proteins between HSC and rMF isolated from normal rat liver. The basis of this
study was the different growth behaviors of activated HSC and rMF.
Although prolonged cultivation of rMF was possible, activated HSC could
be maintained in culture only until passage 2, when the
first passage was performed at the time of full activation (day
7). In contrast to rMF cultures, a significant proliferation measured as increase of cell number could also not be detected in HSC
cultures. Whether the HSC of the hepatic sinusoid increase in number is
controversially discussed (15, 31, 32). However, mitosis
of myofibroblasts of the periportal and perivenous tracta could be
shown (31, 32, 53). We also reported that HSC undergo apoptosis mediated by the CD95/CD95L system when they are
activated and demonstrated that data show both TGF- or TNF-
inhibit apoptosis and DNA synthesis of activated HSC (42,
44). Because survival of myofibroblasts and myofibroblast-like
cells seems to be crucial for development of liver fibrosis in
chronically damaged liver, it was of great interest to study
apoptosis pathways and the effect of two major fibrogenic
cytokines on apoptosis in rMF. For this purpose, we established
a method to isolate myofibroblasts from rat livers.
Although maximum 2% apoptosis was detectable in the rMF
cultures (primary culture day 7, passages 2,
4, and 6), primary cultured HSC showed 18 ± 2% apoptosis at day 7. In subcultivated HSC
cultures, an apoptosis frequency of >80% could be detected.
Because the CD95/CD95L system is shown to be the major pathway for
spontaneous apoptosis of HSC (42), this system was
also investigated for rMF. Studies (6, 36, 45, 50) on
fibroblast cultures of different organs have shown CD95 expression but
conflicting results with regard to CD95L expression and functionality
of the CD95/CD95L system. Lack of spontaneous apoptosis in rMF
also is not due to the lack of CD95R. In fact, compared with activated
HSC (day 7), surprisingly 70% higher expression of CD95
could be observed in the rMF cultures. However, whereas CD95L is
expressed in activated HSC, it is not detectable in rMF. Probably
because of the higher expression of CD95R, triggering of the CD95/CD95L
system using agonistic antibodies at the low amount of 1 ng/ml led to
apoptosis of >95% of cells of the different rMF cultures
within 2 h. A dependency of apoptosis susceptibility and
amount of CD95-expression could also be shown for normal skin
fibroblasts and keloid fibroblasts (34). Maybe because of
the lower amount of CD95R activated, HSC seem to be more resistant to
triggered apoptosis. In the presence of 1 ng/ml CD95-agonistic
antibodies, the apoptosis rate of activated HSC rose from 18 to
37% but was far below that seen in rMF. A concentration of 3 ng/ml was
needed to lead all activated HSC cultures into apoptosis. A
higher susceptibility of rMF to CD95-mediated apoptosis could
also be deduced from the transfection assays, because supernatants of
the CD95L-transfected rMF cultures led to complete apoptosis of
rMF cultures but only to a significant increase of apoptosis
rate in activated HSC. A greater resistance of activated HSC to
CD95-mediated apoptosis could also be shown when regarding time
dependency of apoptosis occurrence. Although the rMF cultures
could be triggered into apoptosis within 2 h, a period of
5-6 h is needed in case of activated HSC. Both dose- and
time-dependent differences in responsiveness to CD95/CD95L-mediated apoptosis might be the result of differences in intracellular apoptotic signaling pathways triggered by CD95 in these two cell populations. This may also be due to differences in antiapoptotic pathways activated in parallel, thus making activated HSC, in part,
refractory to external CD95L. In their paper, Gong et al. (16) demonstrated an increment of the susceptibility to
apoptosis during the transformation of HSC to myofibroblasts,
which contrasts our results. Although the authors described
myofibroblasts as activated HSC, the behavior on apoptosis
induction due to the CD95 receptor and the lack of CD95 ligand
expression more closely resembles our data of rMF than that of
activated HSC raising questions about the origin of their
"myofibroblasts."
TGF- and TNF-
are two key factors involved in many processes of
tissue repair and in the development of fibrogenic disorders. Both
TGF-
and TNF-
inhibit apoptosis of activated HSC
(43, 44). Although TGF-
also effected an
apoptosis inhibition in rMF, TNF-
stimulated
apoptosis. Because the receptor status of HSC and rMF is
similar for TNFR1 and TNFR2, it might be speculated that activated HSC
and rMF also differ in recruitment and reciprocal influences of
apoptosis-regulating pathways. These investigations seem to be
of special importance because they could open valuable insights into
the mechanisms of fibrogenesis and also offer possibilities for
therapeutic approaches. The fact that TNF-
is upregulated in early
phases of models of acute liver damage and that TGF-
is predominant
in late phases of acute liver damage and in chronic liver damages
suggests (together with our in vitro data) that activated HSC may be of
special importance in tissue repair after acute damage, whereas
cytokine situation in chronic liver damage leads to an environment
allowing rMF to grow out and to take over special functions in tissue repair.
Data of the present report strongly suggest that rMF have to be
considered as an additional population of nonparenchymal cells. rMF and
activated HSC differ in occurrence of spontaneous apoptosis due
to different CD95L expression in their susceptibility to CD95-mediated apoptosis and their reaction on TNF- stimulation. Because
rMF and activated HSC could not be delimited by conventional markers until now, and both cell types of the fibroblastic lineage share great
morphologic similarities, in vitro data obtained from prolonged cultured HSC should be examined critically since the cultures used may
consist of rMF grown out from contaminating cells in nonparenchymal
cell cultures (compare Fig. 1, C and D). This
possibility may also be the reason why many published data attributed
to activated HSC demonstrate conflicting results. Furthermore, the
results presented in this paper could possibly be translated to other fields where transdifferentiation of epithelial to mesenchymal cells
has been supposed (54).
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ACKNOWLEDGEMENTS |
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The authors are indebted to A. Herbst and N. Nolte for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft Sonderforschungsbereich 402 Molekulare und Zelluläre Hepatogastroenterologie, project C6.
Address for reprint requests and other correspondence: G. Ramadori, Dept. of Internal Medicine, Section of Gastroenterology and Endocrinology, Georg August University Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany (E-mail: gramado{at}med.uni-goettingen.de).
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.
December 5, 2001;10.1152/ajpgi.00441.2001
Received 15 October 2001; accepted in final form 26 November 2001.
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