Divisions of 1 Gastroenterology, Hepatology, and Nutrition, 2 Pathology, and 3 Developmental Biology, Children's Hospital Research Foundation and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio 45229-3039
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ABSTRACT |
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The urokinase-type plasminogen activator
(uPA) plays a central role in liver repair. Nevertheless, the hepatic
overexpression of uPA results in panlobular injury and neonatal
mortality. Here, we define the molecular mechanisms of liver injury and
explore whether uPA can regulate liver repair independently of
plasminogen. To address the hypothesis that the liver injury in
transgenic mice results from the intracellular activation of
plasminogen by transgene-derived uPA (uPAT), we generated mice that
overexpress uPAT and lack functional plasminogen
(uPAT-Plg). In these mice, loss of plasminogen abolished
the hepatocyte-specific injury and prevented the formation of
regenerative nodules displayed by uPAT littermates. Despite the
increased expression of hepatic uPA, livers of uPAT-Plg
mice were unable to clear necrotic cells and restore normal lobular organization after an acute injury. Notably, high levels of circulating uPA in uPAT-Plg
mice did not prevent the long-term
extrahepatic abnormalities previously associated with plasminogen
deficiency. These data demonstrate that plasminogen directs the
hepatocyte injury induced by uPAT and mediates the reparative
properties of uPA in the liver.
regeneration; urokinase; proliferation; hepatocyte; tissue remodeling
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INTRODUCTION |
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REPARATIVE RESPONSE TO AN injury requires intact cellular proliferation and coordinated tissue remodeling. Shortly after injury, proliferation of undifferentiated and mature cells promptly increases to repopulate the injured tissue and later undergoes downregulation to prevent overgrowth and neoplasia. Meanwhile, tissue remodeling demands restructuring of the extracellular matrix to restore the architectural organization of the healthy tissue. In the liver, remodeling is directed by the combined forces of multiple cell-associated and extracellular proteases. Among serine proteases, plasminogen and plasminogen activators play a central role in liver repair after acute injury. In the absence of plasminogen or plasminogen activators, clearance of necrotic cell debris and matrix components is severely impaired, resulting in defective repair after an acute or a chronic injury (3, 4, 17, 19).
The plasminogen system includes the inactive proenzyme plasminogen, which is proteolytically converted to the active serine protease, plasmin, by either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA). In addition to the traditional role in vascular hemostasis, plasmin and plasminogen activators regulate several biological processes involving proteolysis of the extracellular matrix, such as tissue remodeling, inflammation, tumor invasion, and metastasis (1, 2, 8, 9, 26). Although many of these processes are supported by plasmin-mediated fibrinolysis (6, 7), the defective remodeling of livers of mice lacking plasminogen is independent of fibrin deposition (3). An impairment in liver remodeling that is qualitatively similar is also produced when mice lack uPA or both uPA and tPA but not tPA alone (4).
Despite the key role of uPA in promoting liver repair, hepatic
overexpression of uPA in transgenic mice causes severe liver injury
with an early onset (12). Notably, the injury is specific to hepatocytes and is panlobular, leading to decreased survival of
transgenic mice in the first few days after birth. In this paper, we
explore the molecular mechanism(s) mediating these seemingly opposing
effects of uPA in the liver. On the basis of the role of uPA as an
activator of plasminogen, we hypothesized that the liver injury in
transgenic mice is caused by the coexpression of uPA and plasminogen
within hepatocytes. To address this hypothesis, we generated mutant
mice that simultaneously express the uPA transgene (uPAT) and are
homozygous for a null allele in the endogenous plasminogen gene. We
report that the loss of plasminogen completely abolishes liver injury
in mutant mice. Furthermore, by challenging these mice with a
hepatotoxin, we demonstrate that the overproduction of uPA does not
correct the defective remodeling in Plg mice after acute
liver injury. Together these data demonstrate that plasminogen mediates
the pleiotropic effects of uPA in liver injury and repair.
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MATERIALS AND METHODS |
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Reagents. Liver samples were fixed in 10% formalin from Accra Laboratory (Cleveland, OH), and hepatocyte proliferation was detected using a cell proliferation kit obtained from Amersham Pharmacia Biotech (Piscataway, NJ). CCl4 was purchased from Aldrich (Milwaukee, WI), the sedatives ketamine and xylazine were obtained from Phoenix Pharmaceuticals (St. Joseph, MO), and acepromazine maleate was purchased from Fort Dodge Laboratories (Fort Dodge, IA). Protein concentrations were determined using the Bio-Rad protein assay from Bio-Rad Laboratories (Hercules, CA), and total RNA was isolated using TRIzol reagent (GIBCO-BRL; Manassas, VA). Specific signals were detected by Northern blot analysis and quantified using phosphor screens and an ImageQuant phosphoimager from Molecular Dynamics (Sunnyvale, CA). PCR was performed with a 9600 thermocycler from PerkinElmer (Norwalk, CT). Biochemical markers of liver function were determined by automated enzymatic assay using Vistros Chemistry Systems 950 (Johnson & Johnson, Rochester, NY).
Transgenic and gene-targeted mice.
Heterozygous transgenic mice with albumin promoter/enhancer-driven
overexpression of a uPAT were obtained from Jackson Laboratories (Bar
Harbor, ME) (12). To generate mice overexpressing uPAT without plasminogen (uPAT-Plg), we mated uPAT mice with
Plg
mice of a mixed 129 SvJ/CF-1/NIH Black Swiss
background (6, 7). Offspring were genotyped by PCR using
specific primers for endogenous and targeted alleles for plasminogen
and uPAT by using tail biopsy DNA as the template (7, 13).
Mice of all genotypes were housed in standard facilities and were fed
food and water ad libitum. Survival analysis was performed by examining and weighing mice weekly for the entire duration of the study (58 wk).
Animal protocols were approved by the Institutional Animal Care and Use
Committee of the Children's Hospital Research Foundation (Cincinnati, OH).
Liver injury and morphology.
Urokinase transgene-induced liver injury was assessed visually,
biochemically, and microscopically. At specific postnatal ages, mice
were weighed and anesthetized intramuscularly with 0.1 ml of
ketamine/xylanzine/acepromazine (4:1:1) per 30 g body wt, and
blood samples were collected from the inferior vena cava. These samples
were used to determine levels of albumin (indicator of liver function)
and alanine aminotransferase (indicator of liver injury) using an
automated enzymatic assay with the Vistros Chemistry Systems 950 (27). The anterior lobe of the liver was collected, fixed
in 10% formalin, embedded in paraffin, sectioned, and stained with
hematoxylin and eosin for histological analysis. The remaining liver
was snap frozen in liquid nitrogen and stored at 80°C for future
analysis. For electron microscopy, liver tissues were fixed by
immersion in cold 3% glutaraldehyde prepared in 0.175 M cacodylate
buffer and subsequently postfixed in osmium tetroxide. The fixed livers
were then processed into LX-112 resin by routine techniques, stained
with 2% (vol/vol) uranyl acetate and lead citrate, and examined on
Zeiss 912 or Zeiss 10 transmission electron microscopes.
Northern blot analysis. Total RNA was isolated from frozen liver tissue using TRIzol reagent, and the expression of uPAT and plasminogen was determined as described previously (10, 12, 13). Northern blots were hybridized with [32P]cDNA probes for uPAT and plasminogen simultaneously. The specific signal was quantified using phosphor screens and an ImageQuant phosphoimager.
Zymographic analysis of plasminogen activator. Liver protein extracts were obtained from frozen liver tissue homogenized in buffer containing phosphate-buffered saline (in mM: 137 NaCl, 2.7 KCl, 4.3 Na2HPO4 · 7H2O, and 1.4 KH2PO4), 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 10% protease inhibitor cocktail (Calbiochem, La Jolla, CA). Homogenates were centrifuged at 12,000 g for 15 min at 4°C, and protein concentrations in liver lysates were measured using the Bradford method-based Bio-Rad assay. Plasma was obtained from the supernatants of heparinized blood samples after centrifugation at 9,000 rpm. Liver protein extracts (3.0 µg) and plasma samples (0.1 µl) were solubilized in nonreducing buffer and electrophoresed on SDS-polyacrylamide gel cast with 8% casein and 20 µg/ml plasminogen as described previously (12).
Hepatocyte proliferation. Hepatocellular proliferation after CCl4 administration was measured by the incorporation of bromodeoxyuridine (BrdU) by hepatocytes with the use of the cell proliferation kit (Amersham Pharmacia Biotech) as described previously (27). For each liver sample, the hepatocyte-labeling index (percentage of hepatocytes incorporating BrdU) was calculated by counting BrdU-labeled and unlabeled hepatocytes in 10 high-power fields (~100 hepatocyte nuclei per field) by an investigator unaware of animal genotype.
Statistical analysis. Statistics for survival analysis were performed with Lifetest procedure in SAS for Windows version 8.01. The statistical package does not provide for multiple pairwise comparisons among groups. Such comparisons were carried out separately for each pair of groups using the Lifetest procedure, and the probability values were adjusted manually to account for the multiple comparisons. Values for additional experiments are shown as means ± SD, and statistical significance was assessed by Student's unpaired t-test with the significance level at P < 0.05.
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RESULTS |
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Plasminogen deficiency prevents urokinase-induced liver injury.
The albumin promoter/enhancer-driven overexpression of uPAT causes
liver injury that is apparent immediately after birth and increases
neonatal mortality (12). In normal mice, endogenous uPA
production in the liver is low and is derived from nonparenchymal cells, whereas plasminogen is expressed primarily by hepatocytes (28). To determine whether plasminogen activation by uPAT
within hepatocytes is the mechanism of injury in transgenic mice, we mated uPAT mice with Plg mice. Visual inspection of
livers from littermates revealed the typical pale, white appearance of
uPAT livers at 3 wk of age, followed by the formation of regenerative
nodules that gradually expanded and repopulated the entire liver after
8 wk of age (Fig. 1A). In
contrast, livers of uPAT-Plg
mice appeared normal and
indistinguishable from Plg
mice and control littermates.
To demonstrate that normal-appearing livers of uPAT-Plg
mice had expression of uPAT, we incubated Northern blots containing liver RNA from littermates with radiolabeled cDNA probes for
plasminogen and uPAT transgene simultaneously. Using this approach, we
documented ongoing expression of uPAT in normal-appearing livers
of uPAT-Plg
mice (Fig. 1B). Consistent
with the absence of injury, livers of uPAT-Plg
mice never
developed regenerative nodules and displayed a normal appearance
through adult life, suggesting that plasminogen deficiency prevented
hepatotoxicity induced by uPAT.
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uPA overexpression does not correct the defective remodeling of
Plg livers.
We previously demonstrated (4) that uPA deficiency results
in a defect in liver repair after an acute toxic injury. The defect in
removal of necrotic cells and reorganization of the hepatic
architecture is qualitatively similar to but of a lesser degree than
the one produced by plasminogen deficiency (3). To
determine whether uPA-mediated pathway(s) directs liver repair independently of plasminogen activation, we administered
CCl4 to mice of all genotypes at 2 mo of age, a time when
uPAT-Plg
mice display a prominent overexpression of uPAT.
A single dose of CCl4 causes necroinflammatory injury to
centrilobular hepatocytes due to accumulation of CCl3 and
other highly reactive byproducts (20). Visual inspection
and microscopic analyses of livers demonstrated that the centrilobular
injury induced by CCl4 was not affected by the animal
genotype as supported by the similar centrilobular injury within 2 days
(Fig. 4). At 7 and 14 days, the injury
resolved, and the lobular organization was restored in normal and uPAT
littermates. Although uPAT mice had some residual areas of
transgene-induced injury, the additional toxic insult by
CCl4 did not increase mortality or influence the ability of
the liver to undergo a timely repair. More notable was the severe
impairment in repair of livers from uPAT-Plg
and
Plg
mice at 7 and 14 days, which displayed poor removal
of necrotic cell debris and defective lobular organization. The
appearance and extent of the centrilobular injury was similar in the
livers of all uPAT-Plg
and Plg
mice at 7 and 14 days. uPA overexpression in uPAT-Plg
mice did not
influence liver function or the onset/degree of injury, indicated by
similar plasma levels of albumin and aminotransferase in uPAT and
uPAT-Plg
mice at different time points (data not shown).
Notably, the BrdU labeling of hepatocytes was <0.5% for all genotypes
before injection with CCl4, dramatically increased at 2 days after CCl4 in a similar fashion in all groups, and
returned to low baseline levels at 7 and 14 days (Fig.
5).
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uPA overexpression does not correct extrahepatic consequences of
plasminogen deficiency.
We then explored whether uPA overexpression partially or completely
corrects the extrahepatic phenotype displayed by plasminogen-deficient mice by examining littermates weekly and recording survival in littermates 17 mo of age. We found that long-term survival between uPAT-Plg
and Plg
mice decreased
significantly compared with Plg+ and uPAT littermates
(P < 0.01), with no uPAT-Plg
mice
surviving beyond 13.2 mo (Fig. 7). There
was no significant difference in the survival rate between
Plg
and uPAT-Plg
mice. Furthermore, we
found the extrahepatic manifestations of plasminogen deficiency in
Plg
mice (rectal bleeding, rectal and penile prolapse,
and wasting) in >90% of uPAT-Plg
mice, which became
obvious by 10 mo of age (6). These data demonstrate that
the overexpression of uPA does not reverse the extrahepatic effects of
plasminogen deficiency.
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DISCUSSION |
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Our studies demonstrate that plasminogen directs the uPA-induced
liver injury and repair in vivo. The original findings of a panlobular
liver injury in mice overexpressing uPA runs counter to expectations in
view of the growth-promoting properties of uPA (14, 15,
23). On the basis of the restriction of injury to hepatocytes
and on the well-recognized role of uPA as a potent plasminogen
activator, we hypothesized that intracellular production of the active
protease plasmin by uPAT produced the liver injury. To address this
hypothesis, we superimposed the genetic inactivation of the plasminogen
gene in uPAT transgenic mice. In these mice, plasminogen deficiency
completely abolished liver injury induced by uPAT. Despite high levels
of uPA expression by hepatocytes, loss of plasminogen prevented the
accumulation of multivesicular bodies and disruption of the rough
endoplasmic reticulum within hepatocytes that are typically induced by
uPAT (12). As a consequence, liver morphology remained
normal and did not display formation of regenerative nodules during the
neonatal period or early adulthood. Together, these data conclusively
demonstrate that the intracellular availability of plasminogen to
transgene-encoded uPA within hepatocytes leads to disruption of
intracellular organelles and ultimate cellular injury/death. Moreover,
the absence of nodular growth in uPAT-Plg livers formally
rules out a direct growth-promoting role of uPA in the formation of
regenerative nodules independent of plasminogen and the presence of
injury. Instead, regenerative nodules in uPAT livers develop in
response to the injury (only observed in uPAT livers with normal
plasminogen expression), which creates a microenvironment permissive to
growth of hepatocytes that escape the toxic effect of the transgene
(13). Once the injury is established, uPA probably displays growth-promoting properties, as supported by the timely formation of regenerative nodules in uPAT livers and by defective regeneration after partial hepatectomy in uPA-deficient mice
(23).
Timely cellular proliferation is central to the regenerative response
of the liver to an injury. After partial hepatectomy, hepatocytes and
nonparenchymal cells of liver remnants undergo well-orchestrated rounds
of proliferation to restore liver mass (11, 16). With the
use of this experimental model, uPA has been shown to be needed for the
prompt entry of hepatocytes into the cell cycle, but there appear to be
alternative mechanisms to gradually restore liver mass and maintain
survival in the absence of uPA (23). Despite the apparent
role of uPA as a facilitator of hepatocyte proliferation after partial
hepatectomy (23), we previously found that, after a toxic
injury induced by CCl4, uPA deficiency had no detrimental
effect on hepatocyte proliferation; instead, removal of necrotic cell
debris and lobular reorganization were significantly impaired
(4). The features of this defective repair were similar
(but to a lesser degree) to the outcome in plasminogen-deficient
livers. To directly explore the functional relationship between uPA and
plasminogen in regulation of liver repair, we removed plasminogen from
mice with hepatic overexpression of uPA and determined the ability of
livers to undergo repair after CCl4 injury. Although uPA
and the functional homologue tPA stimulate plasmin-dependent and
-independent angiogenesis in vitro, no previous study directly explored
whether uPA can direct proteolysis during tissue repair independently
of plasminogen activation (5). In our studies using
uPAT-Plg mice challenged with CCl4, the
hepatic overexpression of uPA did not correct the defective liver
repair induced by plasminogen deficiency. The predominant phenotype of
these mice was the inability to remove necrotic cellular debris in a
fashion indistinguishable from the phenotype displayed by
Plg
mice. Therefore, these data do not support an
independent role of uPA in liver repair and place plasminogen as the
central regulatory protease in the removal of necrotic cells and
reorganization of the lobular architecture.
The genetic loss of uPA, tPA, or plasminogen leads to a gradual
multisystem accumulation of fibrin. In extrahepatic tissues, fibrin
deposits result in corneal ulcerations, gastrointestinal bleeding,
rectal prolapse, and a progressive wasting syndrome with decreased life
expectancy in the setting of plasminogen deficiency (6, 18, 21,
22). Notably, despite the increased levels of circulating uPA,
similar multisystem abnormalities and abbreviated long-term survival
developed in uPAT-Plg mice. Together, these data
demonstrate that the presence of plasminogen within intra- and
extracellular environments is required for uPA to induce fibrinolysis
and regulate tissue repair.
In summary, our studies reveal a central role for plasminogen in uPA-induced liver injury and repair. The ability of uPA to induce injury through intracellular activation of plasminogen is particularly important in view of the potential use of uPA in the treatment of hepatic fibrosis by the expression of a nonsecreted form of uPA in the liver (24). Although hepatocytes may be an ideal cellular source for high levels of expression of a uPAT, the intracellular generation of plasmin by uPA may induce widespread injury. Whether activation of plasminogen in the extracellular milieu may efficiently decrease hepatic fibrosis through degradation of matrix components depends on whether plasmin directly or indirectly targets fibrin-unrelated substrates. The identification of these substrates will be critical to understanding the biological impact of plasmin in the clearance of hepatic and extrahepatic matrices.
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ACKNOWLEDGEMENTS |
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We thank Dr. William Balistreri for insightful review of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-55710 (to J. A. Bezerra) and National Heart, Lung, and Blood Institute Grant HL-47826 (to J. L. Degen)
Present address of H. Melin-Aldana: Dept. of Pathology, Children's Memorial Hospital, Chicago, IL 60614.
Address for reprint requests and other correspondence: J. A. Bezerra, Division of Gastroenterology, Hepatology, and Nutrition, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jorge.bezerra{at}cchmc.org).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 13, 2002;10.1152/ajpgi.00336.2002
Received 9 August 2002; accepted in final form 8 November 2002.
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