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Introduction
Hepatic fibrosis is a dynamic and sophisticatedly regulated wound healing response to chronic hepatocellular injury.
This fibrotic process results from the accumulation of extracellular matrix (ECM) including collagen, proteoglycan and
adhesive glycoproteins. Hepatic stellate cells (HSC) are recognized as the primary cellular source of matrix components in
chronic liver disease, and therefore play a critical role in the development and maintenance of liver
fibrosis[1]. In the healthy liver, HSC express a quiescent phenotype and are responsible for the storage of vitamin A located within the subendothelial
space of Disse[2]. During fibrosis, HSC become activated and transform into proliferating fibroblast-like cells which display
increased proliferation and migration, enhanced expression of matrix protein, increased production of matrix metalloproteinases
(MMP) and tissue inhibitors of metalloproteinases (TIMP), all of which lead to replacement by interstitial collagen or scar
matrix[3].
Many studies have demonstrated that in hepatic fibrosis progression, the expression of MMP involved in fibrillar
collagen degradation (eg MMP-1 in humans and MMP-13 in rats) is very limited, whereas the expression of MMP-2 is
markedly increased. Evidence from experimental and clinical studies indicates that MMP-2 expression and upregulated
activity is one of the major causes of liver
fibrosis[4_6]. MMP-2 is constitutively expressed and secreted as a latent zymogen
(pro-MMP-2) which is activated by a membrane-linked process mediated by MT1-MMP and
TIMP-2[7,8]. Once activated, MMP-2 is able to degrade the normal subendothelial matrix, hastening its replacement by fibrillar collagen. The presence of this
newly-formed collagen lattice can further promote HSC
activation[5].
Although the underlying mechanisms remain incompletely understood, accumulating evidence indicates that the
migration of activated HSC from the sinusoidal wall and subsequent movement into regions of injury is a key event for the
development of fibrosis. To migrate, HSC must degrade the subendothelial matrix that is rich in collagen IV, the principal
substrate for MMP-2[9].
Experimental and clinical results indicate that oxidative stress plays critical roles in the activation of HSC and hepatic
fibrogenesis[10]. MMP-2 activation is a critical pathophysiological mechanism relating to the survival of HSC, mediated
during the response to oxidative
stress[11_13]. Reducing oxidative stress by antioxidants could be a potential and effective
therapeutic strategy for prevention and treatment of hepatic fibrosis.
Recently, a major polyphenol of green tea, epigallocate-chin-3-gallate (EGCG), was implicated as the main active ingredient.
EGCG is a potent antioxidant that has attracted considerable attention for its role in preventing oxidative stress-related
diseases including cancers, cardiovascular diseases and
fibrosis[14_16]. The inhibiting effects of EGCG on fibroblasts and
vascular smooth muscle cell MMP-2
expression and activation have been recently
investigated[15,17]. However, to our knowledge, no studies have been done to
investigate these effects on HSC.
To gain further insight into the effects of EGCG on the suppression of hepatic fibrosis, the present study investigated the
effects of EGCG on MMP-2 expression and activation, as well as the
in vitro migration and invasion of rat HSC.
Materials and methods
Reagents EGCG (purity >95%) was purchased from Sigma-Aldrich Chemical Co (St Louis, MO, USA); MMP-2 inhibitor
I (OA-Hy) was obtained from Calbiochem (La Jolla, CA, USA); antibodies against MMP-2 and TIMP-2 were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA, USA) and antibodies against MT1-MMP were obtained from Chemicon (Temecula,
CA, USA).
Culture of HSC Rat HSC were kindly donated by Professor Shi-gang XIONG (Department of Pathology, Keck School of
Medicine, University of Southern California, USA). They were cultured in Dulbecco's minimal essential medium (DMEM;
Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Bio-Whittaker, Walkersville, MD,
USA), 100 IU/mL penicillin and 100 µg/mL
streptomycin. Cultures were incubated at 37
oC in a humidified atmosphere of 5%
CO2 and the medium was changed twice a week. Cell viability was tested by trypan blue exclusion. All experiments were
performed with HSC obtained after 6_15 passages following exposure to serum-free culture medium containing 0.1% bovine
serum albumin for 24 h.
Gelatin zymography HSC
(5.5×104/well) in 6-well plastic dishes were cultured with ConA (20 µg/mL) in the presence or
absence of EGCG at the indicated concentration for 24 h. MMP-2 activity in the conditioned medium of cultured HSC was
analyzed by substrate-gel electrophoresis (zymography) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE; 7.5%) containing 0.1% gelatin (Sigma-Aldrich). Equal volumes of samples of conditioned cell culture medium
were mixed with Laemmli buffer under non-
reducing conditions, loaded onto the gel and separated by electrophoresis. After electrophoresis, SDS was removed by
soaking the gels 3 times for 30 min at room temperature in buffer (50 mmol/L Tris_HCl, pH 8.0, 5 mmol/L
CaCl2, 0.02% NaN3 and 2.5% Triton X-100) and incubated for 24 h at 37
oC with the same buffer lacking Triton X-100. Gels were then stained with
0.1% Coomassie Brilliant Blue R-250 and de-stained until clear bands became evident. Quantitative results of the assays were
obtained by densitometry.
RT-PCR HSC (5.5×104/well) in 6-well plastic dishes were cultured with ConA (20 µg/mL) in the presence or absence of
EGCG at the indicated concentration for 24 h. Total RNA was extracted by the use of TRIzol reagent (Sigma-Aldrich)
according to the manufacturer's instructions. Reverse transcription with oligo (dT) priming was used to generate cDNA from
total RNA (2 µg) extracts. The synthesized cDNA for MMP-2, TIMP-2, MT1-MMP and
b-actin were amplified using specific sets of primers. Rat MMP-2 forward primer was 5' GCT GAT ACT GAC ACT GGT ACT G 3' and reverse primer was 5' CAA TCT
TTT CTG GGA GCT C 3'[18]. Rat MT1-MMP (MMP-14) forward primer was 5' GTA CTA CCG CTT CAA TGA GG 3' and reverse
primer was 5' CAC TGC CAG TAC CAG GAG
3'[19]. Rat TIMP-2 forward primer was 5' ATT TAT CTA CAC GGC CCC 3' and
reverse primer was 5' CAA GAA CCA TCA CTT CTC TTG
3'[20]. b-actin primer was designed based on published cDNA
sequences. The forward primer was 5' TGG GAC GAT ATG GAG AAG AT 3' and the reverse primer was 5' ATT GCC GAT AGT
GAT GAC CT 3'. Each PCR mixture contained the appropriate set of forward and reverse primers (0.2 µmol/L), each dNTP at
0.25 mmol/L, 1.25 U Taq polymerase, and 2.5 mmol/L
MgCl2 in a PCR buffer. The PCR procedure consisted of 28 cycles of
denaturation at 95 oC for 1 min, annealing at 58
oC for 1 min and extension at 72
oC for 1 min, with initial denaturation of sample
cDNA at 95 oC for 3 min and an additional extension period of 10 min after the last cycle. The PCR products were subjected
to 1.5% agarose gel electrophoresis, staining with ethidium bromide and quantitation by densitometry using the Image
Master VDS system and associated software (Pfizer, NY, USA).
Western blotting Cells were washed with cold PBS and lysed by the addition of a lysis buffer containing 1% Nonidet
P-40, 50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 0.1% SDS, and protease inhibitor cocktail (Boehringer Mannhein, Lewes, UK.)
for 20 min at 4 oC. Insoluble materials were removed by centrifugation at 15 000
g for 15 min at 4 oC. The supernatant was
saved and the protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA). Cell
extracts (50 µg/lane) were separated via 10% gel electrophoresis and electroblotted onto polyvinylidene fluoride (PVDF)
membranes. Nonspecific binding sites were blocked by incubating PVDF membranes for 1 h in phosphate-buffered saline
containing 5% low-fat dry milk. Membranes were probed with primary antibodies overnight at 4
oC, followed by the application of a secondary horseradish peroxidase-conjugated second antibody. Blots were developed using an enhanced
chemiluminescence detection system (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) according to the manufacturer's
instructions.
Cellular MT1-MMP activity assay
HSC (1×104/well) in 24-well plastic dishes were cultured with ConA (20 µg/mL) in the
presence or absence of EGCG at the indicated concentration for 24 h. MT1-MMP activity was determined with the use of a
commercial kit (Amersham) according to the manufac-turer's instructions. Briefly, MT1-MMP was extracted from cultured
HSC by extraction buffer and its activity detected through activation of the modified pro detection enzyme and the
subsequent cleavage of its chromogenic peptide subs-trate. The resultant color was read at 405 nm on a microplate
spectrophotometer. The concentration of active MT1-MMP in a sample was determined by interpolation from a standard
curve.
Wound healing assay To study the effects of EGCG on HSC migration, a wound healing assay was performed following
standard methods[21]. Briefly, HSC were seeded in 6-well plates in DMEM containing 10% FBS and grown until 90%
confluent. Cells were then serum starved for 24 h, and a linear wound was created in the confluent monolayer using a 200 µL
pipette tip. Cells were then washed with PBS and diluted in DMEM containing 1% FBS. For the evaluation of `wound
closure' in the different experimental conditions, 5 randomly selected points along each wound were marked and the
horizontal distance of migrating cells from the initial wound was measured after wounding. The cell migration distance was
determined by measuring the width of the wound divided by 2 and by subtracting this value from the initial half-width of the
wound.
Cell invasion assay The invasion assay was performed with Transwell 24-well tissue-culture plates (Costar; Corning, NY,
USA) composed of a polycarbonate membrane containing 8 µm pores. The lower and upper parts of the membrane were
coated with 10 µL of type I collagen (0.5 mg/mL) and 20 µL Matrigel (1 mg/mL; Collaborative Research Inc, Bedford, MA).
HSC were seeded on the inner chamber of the Transwell unit at
2×104 cells in 100 µL serum free medium with or without ConA
(20 µg/mL) supplemented with EGCG or OA-Hy (100 µmol/L). The inner chamber was placed into the outer chamber, which
contained 600 µL of DMEM containing
0.1% FBS and was incubated for 24 h at 37
oC in a CO2 incubator. At the end of the incubation, the cells on the upper surface
of the membrane were completely removed by wiping with a cotton swab. The cells that had invaded the lower surface of the
membrane were fixed with methanol and stained with hematoxylin, and
photographed. Cells from various areas of the lower
surface were counted using a computerized video image analyzing system (Leica Quantimet Q500MC). Each assay was
undertaken in triplicate.
Statistical analyses Results were expressed as the mean±SE of at least 3 separate experiments. Results were analyzed by
one-way analysis of variance (ANOVA) followed by the Student-Neumann-Keuls test. Differences
with P values of <0.05 were considered significant.
Results
EGCG influence on ConA-induced activation of MMP-2
MMP-2 is secreted as inactive zymogen pro-MMP-2 by HSC and
ConA induces pro-MMP-2
activation[22,23]. To investigate the inhibitory effects of EGCG on MMP-2 activation, the release of
MMP-2 in the medium was assessed by gelatin zymography. Zymography of culture media of unstimul-ated HSC revealed a
gelatinolytic band of 72 kDa that represented pro-MMP-2 (Figure 1A). After addition of Con-A to HSC and culture for 24 h,
pro-MMP-2 levels were increased. In addition, less intense bands of 66 kDa were present in zymograms. The 66 kDa band
corresponded to the activated form of MMP-2. EGCG at 5, 10,
25, or 50 µmol/L significantly reduced the ConA-induced
activation of MMP-2 by 23.5%±
6.1%, 59.4%±8.7%, 74.7%±13.5%, and 84.5%±10.6%, respectively (Figure 1B), suggesting that EGCG inhibited pro-MMP-2
activation in a dose-dependent manner. Moreover, the basal secretion of pro-MMP-2 was significantly reduced with EGCG
treatment.
Inhibition of MT1-MMP activity by EGCG
To examine the influence of EGCG on the expression of components of the
MMP-2 activation complex, we assessed MT1-MMP and TIMP-2 expression in ConA-stimulated HSC treated with or without
the indicated concentrations of EGCG. The results showed that the expression of TIMP-2 mRNA and protein were not
affected by ConA (Figure 2A). EGCG had no influence on the level of TIMP-2, as compared with the HSC cultured without
EGCG. Previous studies have suggested that the ConA-induced activation of pro-MMP-2 on the cell surface is mediated by
MT1-MMP[22]. Therefore, the possibility that EGCG could prevent ConA-induced MMP-2 activation by inhibiting
MT1-MMP expression in HSC was further investigated. Expression of MT1-MMP mRNA and protein was increased by
ConA-stimulation. However, EGCG had no influence on the level of MT1-MMP with or without ConA (Figure 2A,2B). However, the
cell-associated MT1-MMP activity was strongly inhibited by treatment of HSC with EGCG in a dose-dependent manner
(Figure 3). These results indicated that EGCG reduced ConA-induced MMP-2 activation through inhibition of MT1-MMP
activity.
Inhibition of pro-MMP-2 expression by EGCG
The possibility of EGCG affecting the amount of pro-MMP-2 expression
in HSC was assessed by RT-PCR and Western blot analyses. Serum-starved HSC were cultured with or without ConA (20
µg/mL) in the presence or absence of EGCG for
24 h. The expression of MMP-2 mRNA and protein was upregulated by ConA stimulation. EGCG alone or in the presence of
ConA markedly reduced MMP-2 mRNA after 24-h incubation period (Figure 4A;
P<0.05). Subsequently, as shown by Western blotting, the quantity of pro-MMP-2 protein could also be significantly reduced by EGCG treatment (Figure 4B;
P<0.05).
EGCG influence on cell migration in the wound healing assay
The wound healing migration assay is an established and
widely used procedure that allows an examination of cell migration in response to an artificial wound produced on a cell
monolayer. Incubation of HSC with 1% FBS produced a marked cell migration in the wound area 24 h after wounding, whereas
wounds treated with EGCG showed dose-dependent delays in wound healing under the same conditions (Figure 5A). The
percentage inhibition of wound closure was evaluated on migration distance. As shown in Figure 5A, compared with 1% FBS
stimulation, EGCG at 5, 10, 25, or 50 µmol/L in the media significantly reduced cell migration distance by 24.5%±7.2%,
37.4%±9.3%, 58.7%±11.8%, and
72.1%±10.3%, respectively (Figure 5B). These results indicate that EGCG inhibited the motility of HSC
in vitro.
EGCG effects on HSC invasiveness To evaluate whether ConA-induced invasion of HSC could be affected by EGCG
treatment, the influence of EGCG on the ability of HSC to invade through reconstituted basement membranes (Matrigel) was
tested. Matrigel is a commercial product extracted from a mouse sarcoma rich in extracellular matrix protein. The major
component is laminin, followed by collagen IV and heparan sulfate proteoglycans. As shown in Figure 6, ConA was found
to stimulate matrix invasion of HSC, but this response was markedly reduced by EGCG, whereas EGCG alone did not affect cell
invasion.
To confirm the role of MMP-2 in the regulation of invasion of HSC, we added the specific MMP-2 inhibitor I (OA-Hy)
which completely abrogated the effects of ConA treatment on the invasion of HSC (Figure 6).
Discussion
MMP-2 expression and upregulated activity are one of the major causes of hepatic fibrosis. Increased MMP-2 activity is
believed to be associated with an increase in destruction of the normal liver architecture, promoting its replacement by
interstitial collagen[4,6]. It is therefore possible that reducing MMP-2 activity of HSC could be a potent therapeutic means for
preventing hepatic fibrogenesis. In the present report, we reveal that EGCG is a strong inhibitor of the expression of
pro-MMP-2 mRNA and protein, and more importantly, of the activation of the secreted MMP-2 in HSC.
MMP-2 is secreted as an inactive proenzyme. Several reports have demonstrated that activation of pro-MMP-2 at the cell
surface through a trimolecular complex that includes
MT1-MMP and tissue inhibitor metalloprotease-2
(TIMP-2). MT1-MMP complexed with TIMP-2 serves as a cell surface receptor for pro-MMP-2 by promoting its pericellular proteolysis and
consequent activation[7,24]. However, excess TIMP-2 was observed to specifically inhibit both MMP-2 gelatinolytic activity
and pro_MMP-2 activation by MT1-MMP[25]. The present findings reveal that EGCG strongly reduces the formation of
active MMP-2 in response to ConA in HSC, in which the levels of activation of pro-MMP-2 were
significantly inhibited by EGCG even at a very low dosage (5
µmol/L). ConA-induced activation of pro-MMP-2 is attributed to
MT1-MMP[22]. Our results demonstrate that ConA can increase the expression level of MT1-MMP mRNA and protein. However, neither mRNA
nor protein expression of MT1-MMP are affected with EGCG treatment in HSC. Although TIMP-2 is considered to be an
inhibitor of MMP-2, expression of this inhibitor was unchanged with EGCG treatment. In contrast, EGCG markedly inhibits
MT1-MMP activity in a dose-dependent manner. These results suggest that the prevention of MMP-2 activation by EGCG
is likely to be mediated by the inhibition of MT1-MMP activity. A similar result in human vascular smooth muscle cells was
reported[15], but the mechanism remains unknown. Previous studies have demonstrated that MMP-2 expression requires the
activation of transcription factor
NF-kB[26]. Since EGCG prevents the activation of
NF-kB[27], its effects on MMP-2 expression
is quite likely achieved through this inhibition.
In response to liver injuries, HSC are activated and accumulate at the site of injury, where they produce an extracellular
matrix leading to characteristic patterns of collagen
deposition[28]. Migration of resident HSC in the space of Disse is
considered important for progression of liver fibrosis because it accounts for increased numbers of activated HSC in areas of
injury[29]. Blockade of HSC migration may represent a potential strategy for therapy of liver
fibrosis[30]. Based on insights from these and other recent works, we were prompted to examine the inhibitory effects of EGCG on HSC migration and
invasion. In the current study, we examined the effects of EGCG on HSC migration measured by wound healing assay. Our
results reveal that, in the presence of EGCG, HSC display a dose-dependent inhibition of wound healing. This observation
suggests that EGCG is capable of inhibiting the motility of HSC. The fact that this function of EGCG dose not occur in
vascular smooth muscle cells[31] suggests a differential response to EGCG based on cell type. Clearly, additional experiments
are necessary to elucidate effects of this antioxidant on cytoskeleton and membrane activity required for HSC migration, as
well as the involved mediators and signal transduction pathway. Finally, and more importantly, in our Transwell invasion
model, we observed that ConA-induced HSC invasion was inhibited by EGCG as efficiently as the MMP-2 specific inhibitor
OA-Hy. This striking effect further supports the potential role of EGCG in control of matrix degradation.
The process of HSC invasion requires the active degradation of environmental barriers including components of the
basement membrane and extracellular
matrix[32]. Activated HSC increase their expression and secretion of the active form of
the MMP-2, which is known to be crucial in the invasion process. Recently, the central role of MMP-2 in the proliferation and
invasiveness of HSC was underlined in a study that documented that collagen type I activation of the discoidin domain
receptor 2 increases HSC proliferation and invasion, a phenomenon that directly reflects the increased expression of active
MMP-2[33]. Furthermore, both the broad spectrum MMP inhibitor GM6001 and OA-Hy completely abrogate the oxidative
stress induced proliferation and invasiveness of
HSC[11]. Therefore, it is clear that the antagonistic effects of EGCG on
MMP-2 expression and activation are involved in the inhibition of HSC invasion.
Oxidative stress may be a common factor in chronic liver diseases of different
etiologies[10]. Many agents have been proposed for the prevention and treatment of fibrosis. However, there is no established therapy for the resolution of hepatic
fibrosis. New clinical approaches need to be developed to improve the efficiency of current treatments. The antioxidant
potential of EGCG is far greater than that of vitamin E and/or
C[34], which might allow it to succeed where other antioxidants
have failed in preventing hepatic fibrosis.
In vitro studies have shown that EGCG exerts anti-fibrogenic effects by decreasing
the synthesis of type I collagen, reducing cell proliferation, and by inducing apoptosis on cultured
HSC[35_37]. Taken together, these findings support the suggestion that EGCG is a promising agent for the treatment of hepatic fibrosis.
Whether EGCG has the same effects in vivo needs to be further investigated.
In summary, our results demonstrate that EGCG strongly inhibits pro-MMP-2 expression as well as the conversion of
pro-MMP-2 into its activated form through the direct inhibition of MT1-MMP activity in cultured HSC. In addition, EGCG can
inhibit HSC migration or invasion through the reconstituted basement membrane. These observations suggest a possible
role for EGCG in the treatment and prevention of hepatic fibrosis.
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