Departments of 1 Pharmacology, 2 Medicine, 3 Pathology, and 4 Pediatrics and Cell Biology, Vanderbilt University Medical Center and Veterans Affairs Medical Center, Nashville, Tennessee 37232
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ABSTRACT |
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To test the hypothesis that
urokinase-type plasminogen activator (uPA) plays an important role in
liver regeneration in vivo, partial hepatectomy was performed on
wild-type and uPA-deficient (uPA/
) mice. Mice were
studied at 24, 44, and 96 h and at 8 days and 4 wk post-partial
hepatectomy for evidence of regeneration, as measured by mitotic
indexes and [3H]thymidine incorporation. In
wild-type mice, thymidine incorporation peaked at 44 h and
this index was reduced by 47% in uPA
/
mice (P = 0.02). By 8 days, however, liver mass was comparable in both groups.
Histological analysis revealed the presence of focal areas of fibrin
deposition and cellular loss by 24 h that were more severe and
prevalent in uPA
/
mice than in wild-type mice (62 and
23%, respectively;
2 = 3.939, P = 0.047). In
contrast, regeneration was not impaired in uPA receptor
(uPAR)-deficient mice at 24 and 44 h. Taken together, these data
indicate that uPA, independent of its interaction with the uPAR, plays
an important role in liver regeneration in vivo.
proteolysis; apoptosis; extracellular matrix; partial hepatectomy; urokinase-type plasminogen activator receptor; DNA synthesis
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INTRODUCTION |
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LIVER REGENERATION IS a complex phenomenon that
involves multiple parallel and sequential processes that are normally
integrated to provide for the timely and complete restoration of liver
mass following injury (7, 18, 35, 42). In addition to the elaboration
of signals to initiate the coordinated proliferation of the various
cellular compartments within the liver, there must be extensive
restructuring of the extracellular matrix (ECM), maintenance of complex
differentiated functions, and a timely restraint of the proliferative
process to prevent overgrowth or neoplasia. Early studies had suggested
the existence of circulating hepatic growth regulators acting through
classic endocrine pathways (19, 37), whereas more recent studies have
proposed that growth factors and cytokines produced within the liver
and acting through paracrine pathways (reviewed in Refs. 7, 18, 35, and
42), are important regulators of liver regeneration.
However, these two modes of regulation are not mutually exclusive: the
nonproliferating liver is a repository of significant amounts of many
of these growth-regulating agents in membrane- or matrix-anchored
latent forms. Proteolytic activation of these proteins to release both endocrine and paracrine factors may be a significant mode of growth regulation during regeneration. In particular, latent or
membrane-anchored forms of hepatocyte growth factor (HGF), transforming
growth factor- (TGF-
), and TGF-
have been shown to be
concentrated in the liver (23, 33, 41).
Considerable circumstantial evidence has suggested that urokinase-type
plasminogen activator (uPA) may play a key role in liver regeneration.
uPA converts plasminogen to plasmin, which in turn promotes matrix
degradation by activating matrix metalloproteinases (MMPs). Urokinase
has the capacity to activate HGF (32, 38) and, via the generation of
plasmin, also activates latent TGF- (20). Therefore, uPA activity
may be required not only to initiate the breakdown of ECM but also to
cleave inactive HGF to initiate hepatocellular proliferation (35) and,
perhaps, to activate TGF-
to restrain proliferation. In support of
this model, uPA activity and the expression of urokinase-type
plasminogen activator receptor (uPAR) increase within 5 min of partial
hepatectomy (PH) in the rat (31). In addition, there is
extensive degradation of ECM (24), generation of plasmin (24), and
increased plasma levels of HGF (29) and TGF-
(34) within minutes of
PH, even though levels of HGF (53) and TGF-
(5) mRNA do not change in the liver or other tissues for several hours. Direct evidence that
uPA is critical for liver regeneration has not been reported, however.
In this study, we test the hypothesis that uPA is necessary for liver
regeneration in vivo by studying hepatic proliferation after PH in
wild-type and uPA-deficient (uPA
/
) mice.
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METHODS |
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Mice. Homozygous uPA-deficient mice, homozygous uPAR-deficient mice, as well as littermate controls were kindly provided by P. Carmeliet (Univ. of Leuven, Belgium) and have been previously described (9, 14). Animals were maintained on a 12:12-h light-dark cycle and fed a normal lab chow diet with ad libitum access to water. All procedures involving animals were performed according to the guidelines set forth by the Animal Use and Care Committee of Vanderbilt University.
Partial hepatectomy. PH (70%) was performed on male mice (27.9 ± 5.2 g) under methoxyflurane anesthesia (Metofane; Pittman-Moore, Chicago, IL) by ligating and removing the left lateral and median lobes, including the gallbladder. Warm isotonic saline (0.5 ml) was deposited into the peritoneum before closure. To control for the diurnal variation in liver DNA synthesis (3), all livers were harvested between 10 AM and 2 PM. To measure hepatocyte labeling index, [3H]thymidine (1 µCi/g body wt; 6 Ci/mmol; NEN, Wilmington, DE) was injected into the peritoneum under light anesthesia 2 h before the animal was killed. Aliquots of liver tissue were then snap frozen in liquid nitrogen or placed in 10% Formalin for paraffin embedding.
[3H]thymidine incorporation. [3H]thymidine incorporation into liver DNA was measured as described previously (40). Briefly, 100 mg of liver tissue were homogenized in 19 volumes of ice-cold 5% TCA followed by three washes in ice-cold 5% TCA. Nucleic acids were hydrolyzed in 1.5 N perchloric acid for 15 min at 90°C. Cleared hydrolysate was then mixed with Bioflour purchased from NEN (Boston, MA), and incorporated [3H]thymidine was counted. DNA content was measured by diphenylamine assay as described by Richards (39). Results are reported as counts per minute (cpm) per milligram of DNA ± SE. All statistical analyses were carried out using GraphPad Prism. Student's t-test was applied, and significance was defined as P < 0.05.
Nuclear labeling index. A labeling index was generated from 6-µm Formalin-fixed, paraffin-embedded sections of liver tissue that were dipped in NTB2 photographic emulsion (Kodak, New Haven, CT) and exposed for at least 6 wk at 4°C. The sections were then developed, fixed, and counterstained with Mayer's hematoxylin. Hepatocyte labeling index (% of hepatocytes) was calculated by counting labeled and unlabeled hepatocyte nuclei in three high-power fields (~300 hepatocyte nuclei/field).
Histology.
Formalin-fixed, paraffin-embedded 6-µm representative sections of
liver tissue from wild-type, uPA/
, and
uPAR
/
mice were stained with hematoxylin and eosin for
general morphology, with Oil Red O for fat, and with Lendrum's stain
for fibrin.
Apoptosis assay. Apoptosis was measured on 6-µm Formalin-fixed, paraffin-embedded sections of liver tissue using the ApopTag Plus Fluorescein in situ detection kit obtained from Oncor (Gaithersburg, MD) according to the manufacturer's directions. Negative controls were performed by substituting water for the transferase enzyme on an adjacent section. Apoptotic nuclei were visualized by fluorescence microscopy.
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RESULTS |
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To test the hypothesis that uPA plays an important role in liver
regeneration in vivo, PH was performed on wild-type and uPA-deficient (uPA/
) mice. Thymidine incorporation into DNA was
measured in groups of wild-type and uPA
/
mice (7
n
14) at 24, 44, and 96 h and at 8 days and 4 wk following PH. Before PH was performed, the rate of
[3H]thymidine
incorporation did not differ between uPA
/
and wild-type mice (data not shown). Consistent with previous reports of liver regeneration in mice (10, 52), DNA synthesis in wild-type mice remained
low at 24 h, peaked at 44 h, and then gradually declined to baseline
between 8 days and 4 wk (Fig.
1A).
uPA
/
mice showed a similar pattern of DNA synthesis.
There was negligible DNA synthesis at 24 h, a peak at 44 h, and a less
rapid decline over the subsequent time points. There was a significant
difference in the rate of DNA synthesis in the two groups, with
[3H]thymidine
incorporation reduced by 47% (P = 0.02) in the uPA
/
mice vs. wild-type mice at 44 h. To
confirm this early reduction in DNA synthesis, a nuclear labeling index
was determined from liver sections exposed to photoemulsion (Fig.
1B). Labeled and unlabeled
hepatocytes were counted, and the percentage of labeled nuclei was
calculated. In wild-type mice, the nuclear labeling index was 1.05 ± 0.28% at 24 h, which peaked at 24.9 ± 2.02% at 44 h and
declined to near baseline by 8 days following PH, paralleling the data
on [3H]thymidine
incorporation. In the uPA
/
mice, however, the peak of
nuclear labeling was decreased by 31%
(P = 0.02) at 44 h, was slightly
decreased at 96 h, and was still elevated at 8 days (224%) compared
with control mice.
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To compare the cumulative extent of liver restoration, liver mass,
expressed as total DNA normalized for body weight, was compared at two
time points (Fig. 2). After PH at 8 or 24 days, no differences were identified between uPA/
and
wild-type mice.
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At baseline, livers from uPA/
mice were histologically
normal and indistinguishable from livers of wild-type mice (data not
shown). In addition to the effects on proliferation, three histological
differences between wild-type and uPA
/
livers were noted.
First, 24 h after PH, all of the wild-type livers contained fine
droplets of microvesicular fat within hepatocytes (Fig.
3, A and
B). Fat was present in some of the
uPA
/
livers at this time point but generally in trace
amounts (Fig. 3, C and
E). The accumulation of fat became
prominent and universal in the uPA
/
mice at 44 h
following PH compared with wild-type mice (Fig.
3G). By 96 h after PH, fat
accumulation was generally resolved in wild-type livers (Fig.
4A);
however, an accumulation of fat was still seen at 96 h post-PH in the
uPA
/
mice (Fig. 4B).
The presence of fat was confirmed by Oil Red O staining (data not
shown). Second, patchy areas of cellular loss, which were not
associated with particular zonal locations, were present in over
one-half of the uPA
/
livers at 24 h (62%) but were seen
in only 23% of wild-type livers at the same time point
(
2 = 3.939, P = 0.047; Fig. 3,
C, E,
and H). There was no inflammatory response associated with the areas of cell loss, which were
characterized by fibrin deposits (Fig.
3H) and cytolysis of cells within
the lesion, as well as nuclear pyknosis in intact cells at the
periphery. Third, a difference was noted in the disappearance of
thickened hepatocyte plates due to regeneration. Thickened hepatocyte
plates were seen in wild-type and uPA
/
livers at 44 h,
although this evidence of hepatocyte regeneration was more prominent in
wild-type livers (Fig. 3, D and
F). At 8 days post-PH, the hepatic
plate architecture was largely normal in wild-type mice (Fig.
4C), but hepatic plates were still
thickened in the uPA
/
livers (Fig. 4D). By 4 wk, hepatic plate
architecture was normal in both wild-type and uPA
/
mice
(Fig. 4, E and
F).
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Because mitogens such as HGF have been shown to protect some cell types
from undergoing programmed cell death (21) and because uPA has been
reported to have mitogenic effects (1, 13), we investigated whether the
areas of cellular loss were associated with apoptosis. Sections of
liver tissue from wild-type and uPA/
mice at different
time points following PH were subjected to in situ labeling to identify
nuclei containing fragmented DNA. Basal liver sections from both
wild-type and uPA
/
mice displayed virtually no apoptotic
cells (data not shown). However, by 24 h post-PH, uPA
/
liver sections contained substantially more apoptotic nuclei compared
with control mice, especially within and surrounding the areas of
patchy cellular loss (Fig. 5). At 44 h
following PH, there were virtually no apoptotic hepatocytes in either
wild-type or uPA
/
liver sections. However, at 96 h
post-PH, apoptotic hepatocyte nuclei were increased in
uPA
/
livers compared with controls. By 8 days following
PH, both wild-type and uPA
/
livers contained virtually no
apoptotic nuclei (data not shown).
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In an attempt to determine the mechanism by which uPA contributes to
hepatic regeneration, groups of wild-type and uPAR/
mice
were subjected to PH. DNA synthesis at 24 and 44 h post-PH was then
determined in wild-type and uPAR
/
mice. No difference was
found in [3H]thymidine
incorporation between wild-type and uPAR
/
mice at 24 h
(12.3 ± 2.5 and 9.9 ± 1.8 cpm/mg DNA,
P = 0.4389) or 44 h following PH
(203.1 ± 52.8 and 329.6 ± 94.9 cpm/mg DNA,
P = 0.2192) (Fig.
6), suggesting that uPA-mediated signaling
events via the uPAR or enhanced activity of uPA may not be important in
the regeneration process. No obvious differences in histology were
noted between wild-type and uPAR
/
mice (data not shown).
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DISCUSSION |
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Proteolysis of ECM and of cell surface proteins can elicit a variety of
rapid and dramatic cellular responses (reviewed in Ref. 51). Among
these responses are ECM remodeling and consequent loss of adhesion and
viability signals, including apoptosis, the exposure of previously
cryptic biological activities in ECM, the activation of immune pathways
and the release of cytokines, the release of soluble or activated
growth factors from latent or membrane-associated precursor forms, and
the direct activation of growth factor receptors. uPA is a soluble
serine protease whose activity is focused at the cell surface by its
interactions with the uPAR (16). The uPAR, in turn, interacts with both
vitronectin and with integrin -chains and has recently been shown to
trigger intracellular signals (15, 25).
We found liver regeneration to be transiently impaired after PH in
uPA/
mice. Peak DNA synthesis, which occurred at 44 h after PH in wild-type mice, was significantly reduced in
uPA
/
mice. Other evidence of delay was noted: the normal
phase of fat accumulation (6), which occurred at 24 h in wild-type
controls, was delayed until 44 h in the uPA
/
mice and was
more prolonged. Despite this early impairment, the liver mass of
uPA
/
mice was completely restored by 8 days, although the
restoration of normal hepatic plate architecture was delayed until 4 wk. In addition to evidence of delayed proliferation, patchy areas of
cellular loss associated with an increase in hepatocyte apoptosis and
fibrin deposition were noted in uPA
/
mice 24 h after PH.
Although we have shown that the absence of uPA impairs hepatocellular
proliferation, others have demonstrated that overexpression of uPA in
the liver is toxic to hepatocytes and leads to subsequent regeneration
(27, 28, 43). Both lines of evidence suggest that a local environment
containing uPA is conducive to the proliferation of liver cells.
Urokinase could contribute to the regenerative process by at least
three different pathways: 1)
uPA-mediated activation of latent growth factors such as HGF or
TGF-, 2) uPA-mediated conversion
of plasminogen to plasmin, leading to increased MMP activity and matrix
remodeling, and 3) uPA-activated
signaling via the uPAR, independent of the proteolytic activity of uPA. The availability of genetically engineered uPA and uPAR
/
mice provides a unique opportunity to determine whether uPA and its receptor are absolutely required for liver regeneration and to begin to
study broader aspects of proteolysis during liver regeneration. uPA
/
mice exhibit normal fertility, growth, and life span
but develop fibrin deposits in the skin and gastrointestinal tract as
they age. In addition, occasional fibrin deposits have been noted in
the hepatic sinusoids (9). These sinusoidal fibrin deposits may be
related to the cellular loss we noted in regenerating liver from
uPA
/
mice, as we rarely noted similar lesions in wild-type controls.
Urokinase is a protease that is known to cleave and activate growth
factors sequestered in the liver. Urokinase activity increases within
minutes of PH in rats (31), whereas the expression of plasminogen
activator inhibitor type I mRNA increases within 2 h (46, 47). uPA has
been shown to activate latent (single chain) HGF in vitro (38), and
Lindroos et al. (29) reported a 17-fold increase in plasma HGF
concentrations within 2 h of PH in the rat, consistent with a role for
uPA in the release and possibly the activation of latent HGF. HGF is a
potent mitogen that has been proposed to play a prominent role in the
regulation of liver growth (45) and regeneration (35). Despite this
circumstantial linkage of uPA activation and HGF release, Miyazawa et
al. (36) failed to detect conversion of HGF to its two-chain form in
the liver after PH, although they did detect activated HGF in liver lysates after CCl4 administration.
Furthermore, exposure of liver to collagenase has been shown to
increase its subsequent mitogenic responsiveness to HGF in the absence
of PH, suggesting that proteolytic alterations in the ECM can also
augment hepatocyte sensitivity to mitogens (30). Our efforts to
directly measure HGF activation in uPA/
mice after PH
have thus far failed to show major differences in HGF activation due to
poor cross-reactivity with mouse HGF (data not shown).
Plasmin generated by uPA in the liver is also likely to result in the
activation of MMPs and of latent TGF-, which increases in the plasma
shortly after PH (34). Recent data from Kim and Michalopoulos (24)
provide evidence of extensive ECM remodeling after PH in rats. They
reported increases in the conversion of plasminogen to plasmin and the
degradation of fibrinogen, fibronectin, entactin, and laminin within
hours of PH, but they did not establish whether plasmin acted directly
on matrix proteins or activated MMPs. Previous studies have shown that
MMPs, perhaps of Ito cell origin (2), are upregulated after PH or
CCl4 injury to the liver (22),
whereas the Ca2+-activated neutral
proteinase is also upregulated after
CCl4 injection (54).
Association of uPA with its receptor, the uPAR, is instrumental in
enhancing its enzymatic activity (17), and the uPAR itself has been
shown to activate cellular signal transduction pathways involving
changes in intracellular tyrosine phosphorylation (4), diacylglycerol
formation (12), and activation of the Janus kinase/signal transducer
and activator of transcription pathway (15, 25). The uPAR itself
contains no tyrosine kinase or transmembrane domains and attaches to
the cell membrane through a glycosyl phosphatidylinositol anchor (11).
It is expressed in hepatocytes, Kupffer cells, and endothelial cells
(26) and is upregulated within minutes of PH (31). In a variety of
different cell types, uPA acts as a mitogen (1, 13) and, by virtue of
its association with the uPAR, mediates cell adhesion and migration
through its interactions with integrins and vitronectin (48-50).
It has been proposed that the uPAR is likely to be responsible for
many, if not all, of the actions of uPA, either by its direct signaling
or its localization and augmentation of the catalytic activity of uPA.
However, uPAR/
mice have a surprisingly mild phenotype.
Despite impaired plasminogen activation in vitro, they exhibit normal
thrombolytic activity and ECM degradation in vivo (14). These results
are mirrored in our observations that uPAR
/
mice do not
exhibit the aberrant regeneration seen in the uPA
/
mice.
Our findings suggest that soluble rather than uPAR-bound uPA plays a
predominant role in regulating liver regeneration. Alternatively, other
proteins may exist that bind, localize, or activate uPA.
Given the myriad of regulatory pathways potentially activated by
proteases, the contributions of uPA to the control of liver regeneration are likely to be complex and multifactorial. Our results
provide no clear evidence that uPA initiates critical signals that lead
to cellular proliferation. Our finding of reduced or delayed
proliferation in uPA/
mice suggests that uPA may play a
permissive but not essential role in normal liver regeneration, perhaps
due to the existence of alternative mechanisms that can compensate in
its absence. It is also unclear whether the effects we observed are
specific for uPA or if activity from a related protease such as
tissue-type plasminogen activator could also play a role. Furthermore,
the regulatory actions of inflammatory cytokines on the uPA system (8,
44) suggest that other models of liver injury may utilize the uPA/uPAR
system in different ways. It is important to note that previous studies
implicating uPA and the uPAR in the control of regeneration have been
carried out in the rat, whereas our studies were performed in mice.
Critical differences between mouse and rat liver regeneration may
complicate the interpretation of our current findings. Elucidation of
the mechanisms by which uPA influences regeneration is likely to
generate models of liver repair in which mechanisms of cellular
proliferation are more closely linked to those of tissue and matrix
remodeling. Moreover, the potential involvement of the plasminogen
activator system in states of disordered repair such as fulminant
hepatic failure, liver fibrosis, or hepatocarcinogenesis warrants
further investigation.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-53804 (W. E. Russell) and National Heart, Lung, and Blood Institute Grant HL-50878 (D. E. Vaughan) and by training grants HL-07751 and HL-07411 (H. T. Roselli) and by a Merit Award from the Dept. of Veteran Affairs Research Service (D. E. Vaughan). D. E. Vaughan is a recipient of a Clinical Investigator Award from the Dept. of Veteran Affairs Research Service.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests: W. E. Russell, Vanderbilt Univ., Division of Pediatric Endocrinology, T-0107 MCN, Nashville, TN 37232-2579.
Received 3 June 1998; accepted in final form 10 September 1998.
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