1 Department of Medicine, Activated, but
not quiescent, hepatic stellate cells (lipocytes) have a high level of
collagen type I and smooth muscle actin (SMA) gene expression.
Therefore, stellate cell activation is a critical step in hepatic
fibrosis. The mechanisms leading to stellate cell activation in vivo
are unknown. The characteristic hepatic oxidative stress cascade
induced in rats by CCl4 markedly stimulated stellate cell entry into S phase, nuclear factor (NF)-
CREB phosphorylation; liver fibrogenesis; c-myb expression
OVERPRODUCTION OF COLLAGEN type
I by activated hepatic stellate cells is a critical step in the
development of liver cirrhosis (14-16, 26). However, the
mechanisms leading to stellate cell activation in vivo remain unclear,
and there is no established treatment for hepatic fibrogenesis (9).
Pentoxifylline is an alkylated xanthine, which is clinically useful for
the treatment of conditions involving defective regional microcirculation (38). Although pentoxifylline, a nonspecific inhibitor
of cyclic nucleotide phosphodiesterases (28), inhibits collagen gene
expression in dermal fibroblasts (4), the mechanisms responsible for
this effect are unknown. Moreover, the mechanisms responsible for the
prevention of hepatic fibrosis by pentoxifylline in yellow
phosphorus-induced hepatocellular necrosis (29) remain to be
determined.
In this context, oxidative stress appears to play an essential role,
through the induction of c-myb and
nuclear factor (NF)- In this study, we analyzed the mechanisms leading to stellate cell
activation in vivo in hepatic injury induced by
CCl4 (3, 31). We found that
oxidative stress results in enhanced NF- Animals. Sprague-Dawley male rats
(50-60 g) each received a single intraperitoneal injection of
CCl4 in mineral oil (1:3, vol/vol)
at a dose of 2 ml/kg body wt (3)
(CCl4, pentoxifylline, and
metabolite 5 groups) or mineral oil only (control group). In addition,
animals received intraperitoneal injections (100 µl) of saline
(control and CCl4 groups), 200 mg/kg pentoxifylline (pentoxifylline group), or 200 mg/kg of the ester
prodrug of pentoxifylline metabolite 5 (metabolite 5 group) at the
following times with respect to mineral oil or
CCl4 administration: Cell
isolation. Stellate cells were
prepared from rats of the experimental groups described above by in
situ perfusion and single-step density Nycodenz gradient (Accurate
Chemical & Scientific, Westbury, NY), as described previously (3, 19,
24). Cells were mixed with 9.5 ml Hanks' containing 3 g/l bovine serum
albumin and 8 ml of 28.7% (wt/vol) Nycodenz in sodium-free Hanks'
buffer. The gradient was generated by placing 6 ml of Hanks' albumin
solution on top of the liver cell mixture in a 50-ml centrifugation
tube. After centrifugation (1,000 g,
4°C, 20 min), cells were aspirated from above the interface, washed
twice in serum-free Dulbecco's modified Eagle's regular glucose
medium, and collected. Stellate cells were identified by
their typical autofluorescence at 328-nm excitation wavelength,
staining of lipid droplets by oil red, and immunohistochemistry
with a monoclonal antibody against desmin (3). Greater than 95% of the
isolated cells were stellate cells (3, 19, 24). In some experiments,
cells were cultured on a collagen type I matrix as described previously
(24) and treated with pentoxifylline (100 µM) or metabolite 5 (100 µM) every day for 6 days. Cells were labeled with 2 µCi
[3H]thymidine
(70-80 Ci/mmol, Amersham). After 3 h of labeling, cells were
harvested and
[3H]thymidine
incorporation into DNA was determined as described (8). Detection of
NIH/3T3 fibroblasts were cultured in Dulbecco's modified Eagle's
medium with 10% fetal calf serum (34) and labeled with [3H]thymidine as
described for stellate cells.
Nuclear
extract
preparation. Nuclei were prepared by a
modification of the procedure described previously (7, 8, 24). Cells
were homogenized in 1 ml of 5% citric acid, 0.5% Nonidet P-40, 10 mM
NaF, and 10 mM Na pyrophosphate with a glass Dounce homogenizer with a
loose-fitting pestle. The homogenized cells were placed above a cushion
consisting of 30% sucrose and 1% citric acid. The nuclei were
precipitated by a 4,000 g
centrifugation at 4°C for 30 min and frozen at Immunohistochemistry. Liver tissue was
immunostained with antisera raised against malondialdehyde-protein
adducts as described previously (7, 11, 19). This antiserum is specific
from malondialdehyde-lysine adducts (3, 20). A phase-contrast microscope was utilized for hematoxylin/eosin staining and
immunohistochemistry with alkaline phosphatase secondary antibodies
(Vector Laboratories). Cytochromes utilized were alkaline phosphatase
with fast green as counterstain (Sigma Chemical). Negative control
samples were processed in parallel under the same conditions, but with
omission of the primary antibody.
Synthesis
of
pentoxifylline
metabolite
5. Metabolite 5 of pentoxifylline
(1-[3-carboxypropyl]-3,7-dimethylxanthine) and its ethyl
ester were synthesized as described elsewhere (12). Briefly, theobromine (2 mmol) was combined with anhydrous
K2CO3
(2.5 mmol) and dry dimethyl formamide (15 ml), and the
mixture was brought to 75°C. The alkyl halide (2.5 mmol) was added
and the mixture was stirred at 75°C for 18 h. The reaction mixture
was cooled, poured into water (125 ml), and extracted with ethyl
acetate (2 × 75 ml). The organic layer was dried over magnesium
sulfate and evaporated to yield a white solid, which was triturated
with ethyl ether. The resulting solid, analytically pure, was purified
further by crystallization. H nuclear magnetic resonance
spectrum, elemental analyses, and exact mass data were consistent with
the assigned structure.
Superoxide
assay. Human neutrophils were isolated
from heparinized normal donor blood by Histopaque density gradient
followed by hypotonic lysis to remove red blood cells. Cells (5 × 106) were suspended in 200 µl
Hanks' balanced salt solution without phenol red and containing
TNF- Statistical
analysis. Results were expressed as
the mean of at least three independent experiments unless stated
otherwise. The Student's t-test was
used to evaluate the differences of the means between groups, with a
P value of <0.05 treated as
significant.
The role of pentoxifylline [a trisubstituted xanthine derivative
(see Fig. 1) that decreases blood
viscosity] on stellate cell activation in vivo was examined. Rats
were treated with carbon tetrachloride, an hepatotoxin that induces
liver lipid peroxidation (3) and stellate cell activation (31).
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
B activity, and c-myb expression. These
changes were prevented by pentoxifylline, which also decreased
CCl4-induced hepatic injury. As
expected, cAMP-mediated phosphorylation of
CREB-Ser133 was induced in vivo in
stellate cells by pentoxifylline but not by its metabolite 5, an
N-1 carboxypropyl derivative, which
lacks phosphodiesterase inhibitory activity. Stellate cell nuclear
extracts from CCl4-treated, but
not from control, animals formed a complex with the critical promoter E
box of the
-SMA gene, which was disrupted by
c-myb antibodies and competed with by
c-myb cognate DNA. Treatment with
pentoxifylline or metabolite 5 prevented the molecular abnormalities
characteristic of stellate cell activation induced by
CCl4. These results suggest that
induction of c-myb plays an important
role in the in vivo activation of stellate cells. Pentoxifylline blocks
stellate cell activation in vivo independently of its inhibitory
effects on phosphodiesterases by interfering with the oxidative stress
cascade and the activation of NF-
B and
c-myb.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
B, in the activation of cultured stellate cells
by collagen type I matrix or transforming growth factor (TGF)-
(24),
suggesting that a similar mechanism may be responsible for stellate
cell activation in vivo. Pentoxifylline inhibits NF-
B activity,
resulting in a decreased expression of the human immunodeficiency
virus-1 long terminal repeat (5), and prevents the
lipopolysaccharide-induced production of tumor necrosis factor
(TNF)-
(25, 32), which in turn activates NF-
B (18). Because
NF-
B activity is induced by oxidative stress signals (18, 24),
resulting in stellate cell activation (24), we tested whether
pentoxifylline prevents the in vivo activation of stellate cells.
B and
c-myb binding activities, which appear
to be critical in stellate cell activation, and that pentoxifylline
blocks these molecular events as well as stellate cell activation.
Moreover, the metabolite 5 of pentoxifylline, an
N-1 carboxypropyl derivative (12, 38) that lacks phosphodiesterase inhibitory activity, also prevented the
activation of stellate cells. These findings suggest that the
inhibition of stellate cell activation by pentoxifylline results from
blocking the oxidative stress cascade within stellate cells, rather
than from its inhibition of phosphodiesterases.
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
4 h,
+8 h, +20 h, +32 h, and +44 h. The last injection (at +44 h) included
30 µCi of
6-[3H]thymidine
(DuPont). Forty-eight hours after the
CCl4 or mineral oil injection (and
4 h after they received
[3H]thymidine),
animals were killed and liver tissues were promptly removed, and a
piece was fixed in 10% formaldehyde and embedded in
paraffin for immunohistochemical staining (3). In some studies, blood
samples were obtained at 24, 36, and 48 h after the
CCl4 or mineral oil injection.
-SMA and cAMP-responsive element binding protein
(CREB)-PSer133 in stellate cell
extracts was performed by Western blot as described (8, 36) and using
antibodies directed against
-SMA (Vector Laboratories) or
CREB-PSer133 (Upstate
Biotechnology).
70°C. DNA
was isolated, extracted, and counted for
[3H]thymidine
incorporation as described previously (8). Gel retardation analysis of
protein-DNA complexes are performed with an oligonucleotide of the
putative DNA binding site, as described previously (7, 8, 24, 37). The
sense oligonucleotides were NF-
B (5'-GGGGACTTTCCC-3')
and
-SMA E box (5'-GATCATAAGCAGCTGAACTGCC-3'). Antibodies directed against c-myb and
NF-
B 65 were obtained from Santa Cruz Biotechnology.
(1 U) and pentoxifylline (100 µM) or metabolite 5 (100 µM).
After 20 min, 1 ml of 120 µM cytochrome c with 100 nM
N-formyl-Met-Leu-Phe (FMLP) was added. After a 10-min incubation at 37°C, the optical density
(OD550) of the supernatant was
determined.
RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References
View larger version (9K):
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Fig. 1.
Structure of pentoxifylline and metabolite 5. Pentoxifylline and
metabolite 5 are trisubstituted xanthine derivatives. Pentoxifylline is
designated chemically as 1-(5-oxohexyl)-3,7-dimethylxanthine, and
metabolite 5 is an N-1 carboxypropyl
derivative of pentoxifylline.
Stellate cell proliferation was assessed by the incorporation of
[3H]thymidine, and
activation by the expression of -SMA (8, 31). The specific activity
of the stellate cell DNA, an index of S phase labeling, was determined
in all groups after 4 h of labeling with
[3H]thymidine at 1:00
PM, to avoid potential variability related to circadian rhythm (8). The
dose of [3H]thymidine
was given intraperitoneally, as described previously (8). The
[3H]thymidine specific
activity of the stellate cell DNA increased ~10-fold 48 h after
administration of CCl4 (Fig.
2A).
Pentoxifylline treatment abolished the proliferation of stellate cells
in animals treated with CCl4 (Fig.
2A), suggesting an inhibitory effect
of pentoxifylline on stellate cell activation. As depicted in Fig. 2B, we found that hepatic stellate
cells of control animals (lane 1) were activated by treatment with
CCl4 (19), as determined by
increased expression of
-SMA on Western blots of freshly isolated stellate cells (lane
2). Treatment with pentoxifylline
prevented activation of stellate cells induced by
CCl4
(lane
3). However, these effects of
pentoxifylline could be the result of interfering with the
hepatocellular injury induced by
CCl4. Therefore, the degrees of
hepatocellular injury and lipid peroxidation were determined in
these animals.
|
Although the histological degree of hepatocellular necrosis (at 48 h) was similar in the CCl4 and CCl4 + pentoxifylline groups, as indicated by liver staining with hematoxylin/eosin (not shown), pentoxifylline reduced significantly (P < 0.05) the release of alanine aminotransferase into the blood at 24, 36, and 48 h (Table 1), indicating a protective effect of pentoxifylline in this animal model of toxic hepatitis. The degree of hepatic lipid peroxidation was comparable in both groups of animals (Fig. 3). Protein adducts with malondialdehyde, a product of lipid peroxidation, were detected using specific antibodies against malondialdehyde-lysine epitopes, as reported previously (3, 19, 20). In contrast to the livers of control animals (Fig. 3A), enhanced lipid peroxidation was comparable at 48 h in zones 2 and 3 of the hepatic acini in animals treated with CCl4 alone (Fig. 3B) or with the addition of pentoxifylline (Fig. 3C).
|
|
Because oxidative stress increases NF-B activity (18) and NF-
B
plays important roles in the regulation of stellate cell activation by
collagen type I and TGF-
(24), we analyzed the potential role of
NF-
B regulation in stellate cell activation in animals treated with
CCl4. Stellate cell activation in
vivo was also associated with the nuclear translocation and activation of NF-
B, as detected by gel shift analysis. As shown in Fig. 4A, the
binding of stellate cell nuclear extracts to a NF-
B cognate oligonucleotide was low in quiescent cells from control animals (lane
2) but increased significantly
following stellate cell activation after treatment with
CCl4
(lane
3). The complex of
32P-labeled NF-
B
oligonucleotides and nuclear extracts from activated stellate cells was
competed with by a NF-
B cognate oligonucleotide (lane
6) but not by unrelated
oligonucleotides (not shown). Of interest, pentoxifylline treatment
prevented not only stellate cell proliferation and activation (Fig. 2 )
but also NF-
B nuclear activity (Fig.
4A,
lane
4) induced by
CCl4.
|
Because c-myb is an important inducer
of proliferation in cultured hematopoietic, smooth muscle, and stellate
cells (1, 24, 33), we tested whether
c-myb expression plays a role in the
activation of stellate cells in vivo. As expected, the critical promoter E box of the -SMA gene (6) formed complexes with nuclear
extracts from freshly isolated activated stellate cells from
CCl4-treated animals (Fig.
4B,
lane
3), but not with nuclear extracts of
freshly isolated quiescent stellate cells from control animals
(lane
2). Treatment of
CCl4 animals with pentoxifylline prevented the formation of a complex between the
-SMA-E box and stellate cell nuclear extracts (lane
4), an essential step in the
activation of the
-SMA gene. Relevant to this study, the protein-DNA
complexes were disrupted by polyclonal
c-myb antibodies without the formation
of a supershift (Fig. 4B,
lane
6), as described for stellate cells
in culture (24), but not by a NF-
B cognate oligonucleotide
(lane
7). Preimmune serum did not affect
the protein-DNA complexes (not shown). These results strongly suggest a
role for c-myb (and presumably an
indirect effect of NF-
B) on
-SMA gene expression, a hallmark of
stellate cell activation (31).
To understand the mechanisms responsible for the blocking effects of pentoxifylline on stellate cell activation, we analyzed whether they are related to its role as a cyclic nucleotide phosphodiesterase inhibitor. A major metabolite of pentoxifylline, metabolite 5 (38), was synthesized as an ethyl ester prodrug (12) (Fig. 1) and found to lack the inhibitory activity of phosphodiesterase from human neutrophils (12).
To test the biological activities of pentoxifylline and metabolite 5 independently of hepatocellular necrosis, we studied their effects on the proliferation of primary rat stellate cells. Both pentoxifylline and metabolite 5 blocked stellate cell proliferation induced by collagen type I matrix (Fig. 5A), which is mediated by an oxidative stress cascade (24). These results indicate that pentoxifylline and metabolite 5 are able to block the oxidative stress cascade induced by collagen type I matrix (24) acting directly on stellate cells. Pentoxifylline and metabolite 5 also inhibited the proliferation of NIH/3T3 fibroblasts induced by serum with a half-maximal inhibitory concentration (~100 µM) (Fig. 5B) similar to that observed in stellate cells.
|
Next, we determined whether pentoxifylline inhibits cAMP phosphodiesterase activity in hepatic stellate cells in vivo, leading to an increase in protein kinase A-mediated phosphorylation. This signal-transduction pathway triggers site-specific phosphorylation of the nuclear transcription factor CREB on Ser133 (17). Therefore, the induction of CREB phosphorylation at Ser133 was analyzed in nuclear extracts from freshly isolated stellate cells using an antibody against the activated, phosphorylated form of CREB (21). As depicted in Fig. 6, treatment with pentoxifylline markedly increased the expression of CREB-PSer133 (43 kDa) (lane 3), which was also detected in stellate cells from control groups in longer exposures (not shown) (21), but not in stellate cells from CCl4 (lane 2) or CCl4 + metabolite 5 (lane 4) groups. In addition to recognizing CREB, anti-CREB-PSer133 detected two other proteins. These are most likely members of the CREB-activating transcription factor (ATF) family, ATF-1 and cAMP response element modulator protein (CREM), that have phosphoacceptor sequences similar to that of CREB-PSer133 (21). Nuclear extracts of stellate cells isolated from control (Fig. 6, lane 1) and CCl4 + metabolite 5 (lane 4) animals also contain small amounts of phosphorylated ATF-1 (38 kDa) and CREM (30 kDa). Neither CREB-PSer133 nor phosphorylated members of the CREB-ATF family were detected in stellate cell nuclear extracts from CCl4-treated animals (lane 2). This study indicates that pentoxifylline, but not metabolite 5, displays cAMP phosphodiesterase inhibitory effects on hepatic stellate cells in vivo.
|
In subsequent experiments, we analyzed the effects of metabolite 5 on
CCl4-induced hepatotoxicity and
stellate cell activation. Treatment with metabolite 5 prevented to a
considerable extent the proliferation (Fig.
2A) and activation (Fig.
2B,
lane
4) of stellate cells induced by the
administration of CCl4. Moreover, treatment of CCl4 animals with
metabolite 5 also prevented the molecular changes characteristic of
stellate cell activation, including enhanced nuclear activities of
NF-B and c-myb (Fig. 4). These
findings suggest that the cyclic nucleotide phosphodiesterase inhibitory activity of pentoxifylline is not indispensable to block
stellate cell activation or proliferation.
Coculture experiments of hepatocytes and stellate cells treated with
CCl4 indicate that hepatocytes
exert a paracrine stimulation of both lipid peroxidation and collagen
gene expression on stellate cells (3). Because pentoxifylline and
metabolite 5 interfere with the oxidative stress cascade in
CCl4-induced hepatic injury (Table
1) as well as in primary rat stellate cells (Fig.
5A), we analyzed whether a similar
effect on oxidative stress occurs in activated neutrophils. Both
pentoxifylline and metabolite 5 prevented the production of superoxide
in human neutrophils treated with TNF- and FMLP (Fig.
7), known inducers of oxidative stress in
these cells (2, 18, 23).
|
The redox state of the cell may alter the DNA binding affinity of
activator protein-1 (AP-1) factors and
c-myb (39, 40). The modulation of
c-myb is probably mediated through a
conserved cysteine amino acid motif (KQC43R) within the DNA binding
domain (40). Therefore, we determined whether 1,4-dithiothreitol (DTT) would normalize the increased binding of stellate cell nuclear extracts
from CCl4-treated animals to the
-SMA-E box. The addition of DTT to the nuclear extracts as described
previously (40) (Fig. 4C,
lane
4) normalized the increased
DNA-protein complex (compare lanes
2 and
3), suggesting that the critical
mechanism involves oxidation of a transcription factor, presumably the
DNA binding domain of c-myb.
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DISCUSSION |
---|
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---|
In this study, we have characterized some of the molecular mechanisms involved in the activation of stellate cells in vivo, an important step in hepatic fibrogenesis (14, 15). Moreover, we have identified a cellular pathway leading to stellate cell activation that is blocked by pentoxifylline. The inhibitory activity of pentoxifylline on cyclic nucleotide phosphodiesterases is not required to block stellate cell activation.
Our results suggest a critical role of NF-B and
c-myb on stellate cell activation in
vivo, given that during stellate cell activation the nuclear activities
of NF-
B and c-myb are increased and
that these molecular changes and stellate cell activation are both
blocked by pentoxifylline and its metabolite 5. Also, we determined
that in activated stellate cells from
CCl4-treated animals,
c-myb contributes substantially to
nuclear binding to the key E box within the promoter of the
-SMA
gene. These findings strongly suggest that
c-myb is the molecular mediator of
oxidative stress on stellate cell activation and that it binds to
the critical E box of the
-SMA gene (6), the expression of which is
intrinsic to the activated phenotype of stellate cells (31).
Pentoxifylline and the metabolite 5 inhibited the enhanced NF-B and
c-myb activities of stellate cells in
CCl4-induced hepatic injury. The
dose of pentoxifylline used was not toxic to the animals, as judged by
their normal behavior, lack of hepatotoxicity, and preservation of
cAMP-mediated phosphorylation of stellate cells. However,
pentoxifylline ameliorated the release of alanine aminotransferase, indicating a protective effect in
CCl4-induced hepatotoxicity. Given
that pentoxifylline inhibits the lipopolysaccharide-induced production
of TNF-
(25, 32), it might inhibit this critical pathway in Kupffer
cells, resulting in decreased leakage of alanine aminotransferase (10).
However, it is improbable that the inhibitory effects of pentoxifylline
and metabolite 5 on stellate cell activation/proliferation in vivo are
mediated by inhibition of TNF-
production. TNF-
has no (or a
small) stimulatory effect on stellate cell proliferation and no effect
on stellate cell activation (9). Finally, collagen gene expression in
stellate cells is inhibited by TNF-
, an effect incongruent with a
potential role of TNF-
in stellate cell activation (9).
Hepatocytes can exert a paracrine stimulation of both lipid
peroxidation and collagen gene expression on stellate cells (3). Potential mediators of this model of oxidative stress in stellate cells
include free radicals, reactive aldehydes, or cytokines produced by
hepatocytes in response to CCl4. In this context, we have
demonstrated the propagation of the oxidative stress cascade to
stellate cells after CCl4
treatment, as judged by activation of NF-B and
c-myb. The increase in this oxidative
stress cascade within stellate cells suggests that this mechanism may
be responsible for the molecular changes leading to stellate cell
activation, as predicted from studies in cultured stellate cells (24).
Several cytokines stimulate stellate cell activation, including
platelet-derived growth factor (PDGF) and TGF-
(30). Interestingly,
the induction of stellate cell activation by TGF-
utilizes an
oxidative pathway, because it can be blocked with antioxidants (24).
Similarly, PDGF requires
H2O2
generation for signal transduction in vascular smooth muscle cells
(35). In addition, Ras-transformed NIH/3T3 fibroblasts produced large
amounts of superoxide, and their mitogenic activity was inhibited with
the antioxidant
N-acetyl-L-cysteine (22). Pentoxifylline also inhibited proliferation of NIH/3T3 fibroblasts. These studies support the notion that cytokine and oxidative stress pathways converge in the activation of stellate cells
(24) and that pentoxifylline blocks these pathways, as suggested for
superoxide production by neutrophils (13).
The binding of c-myb (and other
transcription factors) to cognate DNA sequences can be modulated by the
redox state of the cell (39, 40). We found that the addition of DTT, a
reducing agent, normalized the increased -SMA-E box binding
activities of stellate cell nuclear extracts from
CCl4-treated animals, suggesting an oxidative modification of a critical binding factor, such as c-myb. Future studies should assess
whether this oxidative cascade involves the nuclear redox factor Ref-1
(39), which functions as a DNA repair enzyme and modulates the DNA
binding activity of several transcription factors (40). Although
xanthine and hypoxanthine can initiate the production of
hydroxyperoxide radicals in the presence of xanthine oxidase (2, 23),
they are also capable by themselves of scavenging these free radicals
(2). Given that pentoxifylline and metabolite 5 are substituted
xanthines (12, 38), it is likely that this structure is responsible for
blocking the oxidative stress cascade in the liver of animals treated
with CCl4 as well as in
primary stellate cells. Indeed, we demonstrated that pentoxifylline and
metabolite 5 prevent the formation of superoxide induced by TNF-
and
FMLP, indicating that both act as scavengers of free
radicals.
Increased cAMP-dependent protein kinase activity in stellate cells
treated with pentoxifylline was evident by the phosphorylation of CREB
on Ser133, a classic
phosphoacceptor for protein kinase A (17, 21). This assay also
documented the inability of metabolite 5 to affect phosphodiesterase
activity in vivo. Altogether, these results indicate that the effects
of pentoxifylline on stellate cell activation are independent of its
phosphodiesterase inhibitory activity and are most likely related to
its ability to block the propagation of oxidative stress and the
induction of NF-B and c-myb within the stellate cells. Studies in animals with targeted deletions of
NF-
B or c-myb may establish the
role of these genes in stellate cell activation in vivo.
Our study provides insights into the molecular mechanisms leading to hepatic stellate cell activation in vivo and the blocking effects of pentoxifylline on these pathways. The findings presented here should facilitate potential therapeutic approaches for hepatic fibrosis, a major contributor to the morbidity and mortality of patients with chronic liver diseases.
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to M. Buck for valuable suggestions. We thank K. Pak for technical assistance and L. Masse for the preparation of this manuscript.
![]() |
FOOTNOTES |
---|
This study was supported in part by National Institutes of Health Grants GM-23200, DK-38652, DK-46971, and GM-47165 and by grants from the Department of Veterans Affairs and the American Liver Foundation. K. S. Lee was supported by a grant from Yonsei University College of Medicine (Seoul, South Korea), and K. Houglum was supported by a Clinical Investigator Award (DK-02265).
Address for reprint requests: M. Chojkier, Dept. of Medicine and Center for Molecular Genetics (9111-D), Univ. of California, San Diego, CA 92161.
Received 11 July 1997; accepted in final form 4 August 1997.
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