MINIREVIEW SERIES FOR THE 50TH
VOLUME
Reversibility of hepatic ®brosis: from the ®rst report ofcollagenase in the liver to the possibility of gene therapy
for recovery
Isao Okazaki, Tetsu Watanabe,1 Shigenari Hozawa, Maki Niioka, Masao Arai2 and Katsuya Maruyama3
Department of Community Health, 1Department of Environmental Health, Tokai University School of Medicine
2Department of Internal Medicine, Hiratsuka City Hospital, 3Clinical Research Unit, Kurihama National Hospital
Kanagawa, Japan(Received for publication on February 19, 2001)

Abstract. Since the authors reported the presence of collagenase in the liver as well as its increased
activity in the early stage of hepatic ®brosis and its reduced activity in advanced ®brosis in rats inducedby chronic CCl4 intoxication, in baboons fed alcohol chronically and in patients with alcoholic ®brosis,
other investigators have demonstrated the same tendency of collagenase activity biologically and histochemically. Very recently, the authors demonstrated de®nite gene expression of collagenase during
the recovery from experimental hepatic ®brosis using Northern blotting and in situ hybridization. The®ndings of in situ hybridization not only demonstrated the cells expressing collagenase, but also suggested much information on the mechanism of the recovery from ®brosis. Hepatic stellate cells play a
key role not only in ®brogenesis but also in ®brolysis. The authors' recent observation revealed thatcollagenase (matrix metalloproteinase-13 (MMP-13)) gene expression appears very early in the process of recovery from liver ®brosis, and that both stellate cells and hepatocytes express MMP-13.
Recovery from liver cirrhosis requires the gene expression of collagenase, increased production of thecollagenase enzyme, and activation of the enzyme balanced with the speci®c inhibitors of collagenase.
The understanding of molecular mechanisms of MMP-1 gene expression which is under investigation in
our laboratory may provide us a new strategy for the treatment of liver ®brosis including the possibilityof gene therapy. (Keio J Med 50 (2): 58±65, June 2001)
Key words: collagenase, matrix metalloproteinase (MMP), MMP-13, recovery from liver ®brosis, livercirrhosis

disease, hemochromatosis and other liver diseases.8±10
The authors demonstrated the disappearance ofliver ®brosis during recovery from hepatitis B viruspositive subacute hepatitis with massive ®brosis.11
More recently, treatment with interferon reversed
hepatic ®brosis in patients with hepatitis C virus antibody-positive chronic hepatitis and cirrhosis.12
Recovery from liver ®brosis involves the destructionof newly formed ®brous bands in the liver. Since Grossand Lapiere13 found tadpole collagenase in 1962,
interstitial collagenase has been isolated from skin,uterus, granulocytes, macrophages, and other organs

Introduction
The reversibility of liver ®brosis has been observed
experimentally1±7 and clinically8±12 since Cameron
and Karunaratne1 observed this phenomenon in the
carbon tetrachloride-induced cirrhosis model after removal of the toxic agent. Hepatic ®brosis induced bythioacetamide,2 a-naphthylisothiocyanate,3 ethionine,4choline-de®cient diet5 as well as by the ligation of bileduct6 were demonstrated to be reversible after removalof the causative agent. Recovery from hepatic ®brosis
has been observed among patients with alcoholic liver

Reprint requests to: Dr. Isao Okazaki, Department of Community Health, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa
259-1193, Japan, e-mail: okazaki@is.icc.u-tokai.ac.jp58Keio J Med 2001; 50 (2): 58±65

and cells,14,15 and it has been noted that this enzyme issynthesized de novo and excreted extracellularly, canattack the collagen molecule at three-quarters from theamino terminal end under neutral pH,14,15 and plays
an important role in growth, in¯ammation, tumor
development and other physiological and pathologicalconditions.14,15 The authors previously postulated
the presence of interstitial collagenase in the liver anddemonstrated the ®rst report of collagenase activity
in experimental liver ®brosis.16 This review discusses
the roles of interstitial collagenase in liver ®brogenesisand ®brolysis; recent advances in the understanding ofmatrix metalloproteinases (MMPs) and their speci®ctissue inhibitors of metalloproteinases (TIMPs); andthe possibility of gene therapy causing MMP-1 gene
expression.
The First Report of Collagenase in Experimental
Liver Fibrosis
In 1974 the authors demonstrated collagenolysis
around the explant of a slice of rat ®brotic liver on acollagen gel ®lm, and demonstrated the typical collagenase attack pattern against neutral salt-extracted collagen by disc electrophoresis of the sample collected fromthe reacted collagen gels.16 This disc gel revealed b A(3/4-length of b chain), a A (3/4-length of a chain) anda B (1/4-length of a chain) products, which are the typical products of limited collagen degradation by mammalian collagenase on polyacrylamide gels.
In the following study, the authors used type I collagen that had been puri®ed from acid-soluble collagens
extracted from rabbit skin.17,18 The enzyme source was
the liver of baboons fed an alcohol-containing diet. The
reaction mixture containing type I collagen as a substrate, the homogenate of baboon liver as an enzyme,
and 3mM p-chloromercuribenzoate to inhibit thiol proteinase activity and to convert procollagenase into theactive form, was incubated under neutral pH7,6 at 27 C,
assayed by viscometer, and monitored by disc electrophoresis.17 The levels of the reaction products of b A
and a A and their ratio showed the predicted reaction
pattern by interstitial collagenase. Using this assay
method and the same substrate, the authors demonstrated increased activity of interstitial collagenase in
the early stage of hepatic ®brosis in baboons that had
been fed alcohol chronically17,18 and in patients withalcoholic hepatic ®brosis.18 This research was performed 20 years ago, but the results are still relevant
today. The authors also measured the level of collagenase activity in the liver of rats fed alcohol chronically19,20 and in rats that had been chronically treatedwith carbon tetrachloride.21 However, the level of collagenase activity in these rats was quite low and there
was a slight, but not signi®cant, increase in collagenase59

activity in the early stage of ®brosis. Therefore, theincreased collagenase activity during early ®brosisin these rats had been observed only by a semiquantitative, unspeci®c tissue culture method.21,22
Other researchers subsequently demonstrated thepresence of increased collagenase activity in the earlystage of liver ®brosis and reduced collagenase activity
in advanced ®brosis.23±25
Possible Participation of MMPs and TIMPs
in Extracellular Matrix Metabolism in the Liver
Advances in genetic research have changed themethod of identi®cation of enzymes. For example,
interstitial collagenase was originally de®ned as the
enzyme that can degrade interstitial collagen at a speci®c site under physiological conditions as mentioned
above.14,15 However, recent advances have revealed
that MMP-1, MMP-8 and MMP-13 can also degradeinterstitial collagenase. These MMPs belong to thefamily of MMPs. They can degrade type I, type III andtype X collagens, but cannot degrade other types ofcollagen such as type IV, type V and type VI, nor othercomponents of extracellular matrix such as proteoglycans and glycoproteins. Presently, eighteen MMPs havebeen identi®ed and are grouped into four subclasses
according to their structure and function.26,27 The
activity of MMPs is regulated by several mechanisms
including regulation of gene expression by cytokines or
hormones; extracellular cleavage of the proenzyme tomake the active form of the enzyme; and speci®c inhibition of the active enzyme by endogenous proteins
known as TIMPs. The metabolism of extracellularmatrix is regulated by MMPs in close association with
TIMP-1, 2, 3, and 4.28±31 In the case of rats, sequencehomology analysis revealed that except for human
MMP-13, there is no sequence in rats that shows
more than 90% similarity with the sequence of MMP-1
in humans. The cDNA of rat MMP-1 has not yet
been cloned, and rat interstitial collagenase should
be considered to be the rat homologue of human
MMP-13.32,33
MMPs other than MMP-1, MMP-8 and MMP-13
cannot degrade type I collagen which is very stable, anda net deposition of type I collagen has been observedin progressive hepatic ®brosis.34±41 Arthur, et al.42,43
reported that hepatic stellate cells secrete a neutral
metalloproteinase that can degrade type IV collagen(a component of the basement membrane), and themetalloproteinase that they isolated seems to be
MMP-2. MMP-2, a potent gelatinase, and membrane
type 1 (MT1)-MMP, an activator of MMP-2, can cleave
native type I collagen,44,45 but with less ef®ciency than
MMP-1.44 Interstitial collagenase is a key enzyme
involved in the degradation of ®brosis, and the expres-60

Okazaki I, et al: Reversibility of hepatic ®brosis

sion of interstitial collagenase in the ®brous liver may
be important from the standpoint of ameliorating liver®brosis.
Advanced liver ®brosis is associated with the appearance of perihepatocellular ®brosis, which contributes to the formation of sinusoidal capillarization.
Intralobular shunt vessels between portal vein tributaries and hepatic vein tributaries are formed and there
is no metabolic exchange of the blood with hepatocytes,
leading to the irreversibility of liver ®brosis. MMP-2,
MMP-3 and MMP-9 can cleave type IV collagen. The
authors previously developed an assay method for typeIV collagenase in the liver and demonstrated reduced
activity of type IV collagenase in human liver cirrhosis.46±48 Generally, however, in the process of woundhealing, type V- and type IV-collagens appear ®rst, followed by the appearance of large amounts of type IIIand type I-collagens. The authors observed reduced
activity of type IV collagenase in liver cirrhosis asmentioned above;46±48 however, the most stable andmost abundant collagen is type I collagen. Therefore,
the authors has investigated the MMP-1/MMP-13 gene
expression in the process of liver cirrhosis.
Gene Expression of MMPs and TIMPs in the
Progression of Liver Fibrosis
The increased collagenase activity in the early stageof liver ®brosis, which had been observed by theauthors16±22 and others,23±25 initially could not be correlated with the results of gene research. For example,
Iredale, et al.28,49,50 did not observe an increase in
MMP-13 mRNA transcription in the liver. Instead, they
demonstrated an increase in TIMPs mRNA transcripts
and postulated that the balance between the downregulation of MMP-13 expression and the upregulationof the expression of TIMPs may result in the depositionof type I collagen in experimental hepatic ®brosis.
The question is which MMPs are responsible for theincreased collagenase activity in the early stage of liver®brosis.
Takahara, et al.51 showed that the level of MMP-2
expression increased during the process of experimental hepatic ®brosis as well as during the process of hepatic ®brosis in chronic hepatitis, and that it decreased
during the process of cirrhosis. Takahara, et al.52 also
demonstrated the dual expression of MMP-2 and MT1MMP in chronic hepatitis and cirrhosis, and further
demonstrated cytoplasmic and membranous immunodeposits of both MMPs in endothelial cells, Kupffer
cells, capillary endothelial cells and lymphocytes. In
particular, they observed the over-expression of MMPs
in stellate cells and ®broblasts, and suggested that MT1MMP activates Pro-MMP-2 and the activated MMP-2
may remodel the liver parenchyma during the processof liver ®brosis. Thus, the gene expression of MMP-2
increased during the process of liver ®brosis anddecreased with cirrhosis. MMP-2 expression is stimulated by transforming growth factor (TGF)-b, and
MMP-2 expression is regulated in a different mannerfrom the expression of interstitial collagenase. TGF-b
upregulates MMP-2 expression while it downregulates
MMP-1 expression. Therefore, the increased activity ofcollagenase in the early stage of liver ®brosis has been
considered to be mediated by MMP-2 and MT1-MMP.
Recently, the authors demonstrated the gene expression of MMP-13 in the CCl4 -treated rat liver by
reverse transcription-polymerase chain reaction (RTPCR) and in situ hybridization.53 In the normal rat
liver, no signal for MMP-13 mRNA was observed by insitu hybridization. In the liver of rats with fatty change
induced by 4-week CCl4 treatment, positive signals for
MMP-13 mRNA were observed in scattered cells, andthese cells were negative for a-smooth muscle actin but
seemed to be hepatic stellate cells because they werepositively stained with desmin. In the rats treated with
CCl4 for 8 weeks, signals for MMP-13 mRNA wereobserved in a few cells lining the ®brous septa. Someof these cells were stained with a-smooth muscle actin
antibody. On the other hand, the cirrhotic liver of rats
treated with CCl4 for 12 weeks revealed very weak
expression of MMP-13 mRNA in stellate cells. No
hepatocyte in the liver revealed MMP-13 mRNA transcripts regardless of the length of CCl4 treatment.
As mentioned above, generally there was very weak
MMP-13 gene expression during the process of rat
hepatic ®brosis induced by chronic CCl4 intoxication.
The authors, however, postulated that an increase in
MMP-13 gene expression, even if the increase is veryslight, may be necessary for the destruction of the tissue
in order to deposit newly formed matrix. Liver ®brosis
in baboons and humans shows a prominent increasein MMP-1 biological activity at the early stage. In fact,
Iredale, et al. reported that the activated stellate cells in
autoimmune chronic active hepatitis produced matrix
components as well as MMP-1.49 The gene expressionof MMP-1/MMP-13 appears in the early stage of liver®brosis before the deposition of ®brosis in the liver.53
Therefore, the increased collagenase activity that theauthors reported in 197416 seems to be mediated by
MMP-13 as well as MMP-2 and MT1-MMP.
Gene Expression of MMPs and TIMPs in Recovery
from Liver Fibrosis
The authors demonstrated that MMP-13 expression
was markedly enhanced during the recovery phase ofliver ®brosis, and cells strongly positive for MMP-13
were observed mainly at the interface between the
resolving ®brous septa and the parenchyma by in situ

Keio J Med 2001; 50 (2): 58±65

61Fig. 2 The gene expression levels of MMPs and TIMPs in theaggressive phase of liver ®brosis (left) and its recovery phase (right).
This ®gure is presented by the permission of the Japanese Society of
Hepatology (originally appeared in Acta Hepatol Jap 2000; 41: 741±
753).

Fig. 1 In situ hybridization of MMP-13 mRNA in rat liver sections
obtained 5 days after 8-week CCl4 treatment. Increased MMP-13
gene expression was observed with the antisense (AS) probe but notwith the sense (S) probe in a serial section. MMP-13-positive cells
were located along the border between the resolving ®brous band andthe parenchyma. Original magni®cation: AS, S 50.

hybridization (Fig. 1).53 Overlapping the images of insitu hybridization and immunohistochemical staining
with the help of a computer revealed that some, but notall, of the MMP-13-positive cells were stellate cells thatwere stained with a-smooth muscle actin antibody. This
was the ®rst report that provided direct evidence ofde®nite MMP-13 gene expression during the recovery
phase, which is in contrast with the downregulation of
MMP-13 expression during the progression of ®brosis(Fig. 2).53
Takahara, et al.51 showed that following the discontinuation of chronic CCl4 treatment, there wasincreased MMP-2 expression on day 3 and day 7, andreduced expression on day 14, and suggested that thedestruction of pericellular ®brosis may occur during
the very early stage of recovery. In situ hybridization
for MMP-254 revealed that vimentin-positive, CD-68negative mesenchymal cells, which are assumed to bestellate cells, showed high transcript levels of TGF-b aswell as MMP-2. Iredale, et al.28 and Roeb, et al.30
demonstrated that the TIMP-1 mRNA level increased
during the early phase of CCl4 treatment, and thendecreased and remained at a low level during the recovery from liver ®brosis. The net activity of MMPs isdetermined by the balance between the activities of the
MMPs and their inhibitors. Herbst, et al.31 revealed
that high levels of TIMP-1 and TIMP-2 transcripts were
present in all ®brotic rat and human livers, predominantly in stellate cells. Iredale, et al.50 reported that thelevels of TIMPs decreased during the recovery phase ofexperimental hepatic ®brosis.
Although the reports of Vyas, et al.,55 Herbst, et al.56
and Winwood, et al.57 showed that MMP-3 and MMP-9
are involved in perihepatocellular ®brosis, there has
been no study on the gene expression of MT1-MMP
and other MMPs in the context of ®brolysis in the liver.
The authors initially had considered that MMP-2 andMT1-MMP are not involved in the recovery from liver®brosis because both MMP-2 and MT1-MMP gene expression decreased during the process of ®brolysis in
the liver. Against our expectation, however, recent ourobservation suggests that both MMP-2 and MT1-MMP
have a some role in ®brolysis although their gene expression decrease in the recovery phase (submitted).
The hepatic stellate cell is a pivotal key player that
produces extracellular matrix, secretes and deposits
matrix with MMP-2 and MT1-MMP extracellularly, andsometimes secretes MMP-1/MMP-13 for ®brolysis after
receiving the signal transduction following removal of
the toxic reagent. The authors' observations led to thenext hypotheses: 1) MMP-13 gene expression appears
very early in the process of recovery, and both stellate
cells and hepatocytes probably express MMP-13 at thisstage. 2) The expression of MMP-2, MT1-MMP and62

Okazaki I, et al: Reversibility of hepatic ®brosis

TIMPs are down-regulated during the recovery fromliver ®brosis, but the cells that are expressing MMP-2,
MT1-MMP or TIMPs change very quickly. Although
the precise mechanism is not known, the co-ordination
among MMP-13, MMP-2, MT1-MMP and TIMPs may
participate in the destruction of newly formed ®brous
tissue in the recovery phase of liver ®brosis.
Induction of MMP-1/MMP-13 Gene Expression
in the Liver
The authors previously investigated the mechanismof collagenase production in liver cells using a monolayer culture of ®broblasts derived from the rabbit
liver.58±60 Different mechanisms of collagenase production were observed among ®broblasts derived fromthe synovium, gastric mucosa and liver of the samerabbit. All ®broblasts used in this experiment were thefourth passaged cells in order to exclude macrophages
and to obtain uniform cell lines. Synovial ®broblasts
secreted a low level of collagenase without any treatment, and those treated with phorbol myristate acetate(PMA) produced a high level of collagenase. Gastricmucosal ®broblasts produced a high level of collagenase
without any treatment. Upon treatment with PMA,
MMP-1 production dramatically increased. Liver ®broblasts did not produce collagenase, even with PMA
treatment.60
The authors succeeded in inducing MMP-1 expression by co-culture of ®broblasts and hepatocytes at thecell number ratio of 3 : 1. After a long latent period, aremarkably high level of collagenase synthesis wasobserved.59,60 The large quantity of collagenase produced by ®broblasts contributes to massive necrosis
or tissue breakdown in vivo. As hepatic stellate cells
can express high levels of MMP-1/MMP-13, MMP-1/
MMP-13 production should be considered to be related
to the activation of stellate cells.
Electron microscopic studies and culture studies ofstellate cells revealed that myo®broblasts are cells that
had been transformed from stellate cells, and may produce extracellular matrix.34±41 This transformation isreferred to as the activation of stellate cells. Stellate
cells are activated by expressing c-myb and NFkB,
which is induced by oxidative stress. This is evidenced
by the following observations. Lee, et al.61 demonstrated that stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and bymalondialdehyde, a product of lipid peroxidation. Stellate cells were also activated by the addition of type I
collagen or TGF-a. This activation was inhibited by anantioxidant (1-a-tocopherol) or butyrate hydroxytoluene. Their valuable ®ndings were that oxidative
stress, TGF-a and collagen type I each cause theproliferation of activated stellate cells by markedly

stimulating NFkB activity and c-myb expression. Antioxidants and c-myb antisense oligonucleotide inhibitedthe activation of stellate cells by type I collagen and
TGF-a; the expression of NFkB and c-myb; and theproliferation of activated stellate cells (myo®broblasts).61 Moreover, they demonstrated that nuclear
extracts from activated stellate cells formed a complex
with the promoter E box of the a-smooth muscle actin
gene, and this process was disrupted by antibodies
against NFkB65 and c-myb. C-myb was also expressedin activated stellate cells in the ®brotic liver of rats that
had been treated with chronic administration of carbon
tetrachloride intoxication.61
Endothelin induces the activation of stellate cells,
and an antagonist of endothelin (bosentan) inhibitedthe activation of stellate cells.62 Platelet-derived growth
factor (PDGF), a cytokine, stimulates collagen production, and pentoxifylline is now known to inhibit PDGF
as well as the activation of stellate cells.63 A recent
study by Marra, et al.64 clari®ed that phosphatidylinositol 3-kinase is involved in the PDGF-induced activation of stellate cells, and that this phosphorylation isnecessary for the motility, proliferation and transformation of stellate cells to myo®broblasts.
The relationship between these activation processes
mentioned above and the mechanism of productionof MMP-1/MMP-13 has not been found. Activatedstellate cells producing extracellular matrix seem not to
express MMP-1/MMP-13 from the authors' observation.53 Therefore, the mechanism of MMP-1/MMP-13
gene expression in stellate cell should be investigated.
The molecular mechanism of the transcriptional regulation of the MMP-1/MMP-13 gene has not beenstudied in hepatic stellate cells. In¯ammatory cytokines
such as tumor necrosis factor (TNF)-a and interleukin(IL)-1b are known to be involved in the activation ofhepatic stellate cells. Most such cytokines activate theretrovirus-associated DNA-mitogen activated protein
kinase (Ras-MAPK) signaling pathway including c-Jun
NH2-terminal kinase (JNK) and extracellular signalregulated kinase (ERK), which in turn activate thetranscription of early genes such as c-fos and c-jun. The
Fos and Jun proteins contribute to the induction of
MMP-1 gene transcription by their binding to proximalAP-1 sites of the promoter in ®broblasts and immortalized cells.65±67 In addition to the role of AP-1, Vincenti,
et al.68 recently reported the role of NF-kB in the induction of rabbit MMP-1 expression in IL1-treatedsynovial ®broblasts. TNF-a and IL-1b increase MAPK
activity including JNK and ERK in rat HSCs, thereby
stimulating AP-1 activity in the hepatic stellate cells.69
Activation of stellate cells is also closely related to NFkB activity.70 Activation of transcriptional factors suchas AP-1 and NF-kB activity may also contribute to gene
regulation of MMP-1 in HCCs. The molecular mecha-

Keio J Med 2001; 50 (2): 58±65

nisms of the regulation of MMP-1 expression are still
unknown. It should be determined how such transcriptional factors as NF-kB or AP-1 may be involved in
MMP-1 gene expression in stellate cells.
Possibility of Gene Therapy to Induce MMP-1 Gene
Expression in Cirrhotic Liver
The recent topic is hepatocyte growth factor (HGF),
that is, the ®brolytic effect of HGF on experimental
liver ®brosis.71±72 Ueki, et al.72 showed that in dimethylnitrosamine-induced liver ®brosis, HGF downregulated TGFb expression, which was followed by thedisappearance of pre-deposited ®brous tissue. Ozaki, etal.73 proposed that HGF induces MMP-1 expression in
LI 90 (cell line of human stellate cells) via Ets-1 in the
promoter region of MMP-1. The recovery from liver®brosis by external HGF treatment, indicates that®brolysis induced by increased collagenase activity
could be related to regeneration of the liver.
The participation of hepatocytes in the destruction of
the extracellular matrix should be considered. The
authors previously reported that transient expression ofinterstitial collagenase was observed in differentiated,very early hepatocellular carcinomas (less than 2 cm),
but not in moderately and undifferentiated hepatocellular carcinomas.74 From the results of our experiments,53,74 we hypothesize that in the recovery phaseof liver ®brosis, liver regeneration occurs through theproliferation of both mature pre-existing hepatocytes
and stem cells, which may express MMP-1/MMP-13
in response to the elevated HGF level to destroy
the extracellular matrix to complete the tissue repair.
MMP-1/MMP-13 transcription is transient, and stem
cells may differentiate into hepatocytes, stellate cells
and other cells. The authors are now investigating thepossibility of gene therapy to proliferate stem cells in
the liver and to express MMP-1/MMP-13 gene in stem
cells with or without transfusion of stem cells derived
from bone marrow in the patients with liver cirrhosis.
Conclusion
The authors believe that MMP-1/MMP-13 can degrade collagen ®bers and contribute to the recovery ofliver ®brosis. Therefore, the gene expression of MMP1/MMP-13 should be a key step in this process. Finally,the ®nding of collagenase expression in the recovery
phase of experimental liver ®brosis by in situ hybridization as well as demonstration of the enzymatic
activity of collagenase indicates that there is promisingpotential for the development of a new therapy whichshould focus on the gene expression of MMP-1/
MMP-13, for the treatment of liver ®brosis, although wemust be careful about tumor development.

63

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