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From the Divisions of Gastroenterology and Nutrition,*
Pathology,
and Developmental
Biology,§
Children’s Hospital Research
Foundation and Department of Pediatrics, University of Cincinnati,
Cincinnati, Ohio; and the Oral and Pharyngeal Cancer
Branch,
National Institute of Dental and
Craniofacial Research, National Institutes of Health,
Bethesda, Maryland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The conversion of the inactive proenzyme plasminogen to the active serine protease, plasmin, by either urokinase-type plasminogen activator (uPA) or tissue-type plasminogen activator (tPA), is critical to the maintenance of hemostasis. Both plasminogen- and plasminogen activator-deficient mice experience widespread microvascular thrombosis, progressive organ damage, and delayed wound healing.11-15 These adverse pleiotropic effects seem to result from the multisystem accumulation of fibrin, as indicated by the general correction of the spontaneous phenotypic abnormalities observed in plasminogen-deficient mice when a loss of fibrinogen is genetically superimposed.16 However, a number of observations support a broader role for the plasminogen activation system outside fibrinolysis,17-19 particularly in the hepatic microenvironment.20-25 Perhaps the most persuasive of these observations is that plasminogen-deficient mice are unable to reorganize hepatic matrix and/or remove necrotic hepatocytes from sites of acute liver injury, regardless of the presence or absence of circulating fibrinogen.10 Interestingly, poor removal of necrotic cells has also been reported in the heart of mice lacking uPA; necrotic cardiomyocytes within ischemic heart tissue persist as mummified ghosts, and are not readily replaced by fibrotic scar tissue in uPA-deficient mice.26 Together, these studies suggest that plasminogen- and uPA-mediated clearance of necrotic cells may be central to tissue repair.
In the current studies, we explored the role of uPA and its functional homologue tPA in liver cell proliferation, matrix remodeling, and clearance of necrotic cells in vivo. Using a well-established model of acute liver injury in mice genetically engineered to lack tPA (tPAo), uPA (uPAo), or both (tPAo/uPAo),12 we report that although cellular proliferation is not affected by the lack of plasminogen activators, repair is severely impaired. Mice lacking each plasminogen activator individually have a mild (tPAo mice) to moderate (uPAo mice) impairment in repair, whereas the combined loss of uPA and tPA results in severe defect in lobular reorganization with matrix accumulation and poor clearance of necrotic hepatocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice with a targeted disruption of the genes coding for tPA (tPAo) or uPA (uPAo) were of a mixed 129/C57BL/6 genetic background.12 To generate mice with simultaneous deficiency of tPA and uPA (tPAo/uPAo), we bred double heterozygous mice, and offspring were genotyped by polymerase chain reaction using specific primers that identify endogenous and targeted alleles for the tPA and uPA genes using ear biopsy DNA as template.12,27 All experimental challenges were performed in 2- to 6-month-old littermates. All mice were housed together, fed laboratory chow ad libitum, and were kept in the same room under supervision by the same investigator throughout the study period. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Children’s Hospital Research Foundation (Cincinnati, OH).
Liver Injury
Gene-targeted mice and control littermates were injected intraperitoneally with 0.5 ml of carbon tetrachloride (CCl4) (Aldrich Chemical Inc., Milwaukee, WI) per kg of body weight as a 25% solution in corn oil.28,29 Mice were examined daily and sacrificed 2 to 35 days after CCl4 treatment, as previously described.10 In brief, mice were weighed, anesthetized, and blood samples were collected from the inferior vena cava. The liver was removed, blot dried, and weighed; samples were obtained from lobes, embedded in paraffin, sectioned, and stained with hematoxylin and eosin for histological analysis. The remainder of the liver was immediately frozen in liquid nitrogen. Biochemical markers of liver function and injury were determined in plasma within 4 hours of collection by an automated enzymatic assay using the Vistros Chemistry Systems 950 (Johnson & Johnson, Rochester, NY).29
Hepatocyte Proliferation
The proliferative response after CCl4
injection was measured by the incorporation of bromodeoxyuridine
(BrdU), which was administered intraperitoneally to all mice 2 hours
before sacrifice.10
BrdU-labeled hepatocytes were
identified on 4-µm sections of paraffin-embedded liver samples
according to instructions provided in the Cell Proliferation kit
(Amersham, Life Science, Arlington Heights, IL).29
For
each liver sample, hepatocyte labeling index (% of hepatocytes
incorporating BrdU) was calculated by counting BrdU-labeled and
-unlabeled hepatocytes in 10 high-power fields (
100 hepatocyte
nuclei/field) by an investigator unaware of animal genotype. Hepatocyte
proliferation was then expressed as the mean (±SD) for all mice in
each group, and statistical significance was determined using unpaired
t-test, with a significance level of P <
0.05.
Plasminogen Activator Zymography
Liver extracts were prepared from frozen liver samples homogenized in phosphate-buffered saline containing 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate, and 10% protease inhibitor cocktail (Sigma Chemical Co., St. Louis, MO). Homogenates were centrifuged at 12,000 x g, for 15 minutes, at 4°C, and protein concentration was determined in the supernatant using the Bradford method-based Bio-Rad assay (Bio-Rad Lab., Inc., Hercules, CA). One hundred µg of homogenate protein were analyzed by zymography using polyacrylamide gels cast with casein and plasminogen as described previously.11,30
Staining of Extracellular Matrix Substrates
Immunohistochemical detection of fibrinogen and fibronectin (two components of the provisional matrix of wound fields) was performed in liver sections using a rabbit anti-fibrinogen antiserum,10 rabbit anti-fibronectin IgG (Sigma Chemical Co.), and the Vectastain ABC-AP detection system (Vector Laboratories, Burlingame, CA); omission of primary antibodies was used in at least one section for each experiment as a negative control.15 Fast Red TR/naphthol AS-MX (Sigma Chemical Co.) was used to detect alkaline phosphatase activity in situ.10
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CCl4 is a specific hepatotoxin that destroys
a subset of hepatocytes proximal to the central vein that enzymatically
convert CCl4 to the free radical
CCl3 and other highly-reactive
species.31
To explore the role of plasminogen activators
in liver repair after an acute toxic injury, mice with single and
combined deficits in tPA and uPA were challenged with a single dose of
CCl4. Control
(tPA+/uPA+) and plasminogen
activator-deficient animals displayed a similar susceptibility to
CCl4-induced liver damage. Plasma alanine
aminotransferase (ALT, an indicator of hepatocyte injury) levels
increased >80-fold within 2 days of the initial challenge and returned
to near baseline by 7 days, regardless of the genotype (Figure 1)
. Therefore, deficiency of plasminogen
activators did not seem to affect either the initial extent of liver
damage or the short-lived toxicity of CCl4.
Consistent with the ALT data, visual inspection of livers 2 days after
CCl4 revealed a similar diffuse pale/lacy
appearance in mice of all four genotypes (Figure 2)
. The gross appearance of the livers of
mice expressing both plasminogen activators
(tPA+/uPA+) or expressing
solely uPA (ie, tPAo) normalized by 7 days. In
contrast, the diffuse pale/lacy appearance persisted in the livers of
both uPAo and
tPAo/uPAo mice, with only
marginal improvement in the former at 14 days. To further define the
long-term outcome of hepatic repair, we further carried the study to 35
days. At this time, the livers of
tPA+/uPA+,
tPAo, and uPAo mice
invariably displayed a normal appearance, whereas the livers of
tPAo/uPAo mice remained
grossly abnormal, and similar to the appearance that had been seen 2
days after CCl4 (data not shown).
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. There was
minimal inflammation and intact cellular components in the unaffected
areas of the liver lobule; venular, sinusoidal endothelial, and
nonparenchymal cells were well preserved. Systematic analysis of liver
samples at 7 to 35 days (n = 3 to 4 for each
genotype) showed that the centrilobular injury of
tPA+/uPA+ mice resolved at
7 days, whereas tPAo livers had minimal residual
necrotic hepatocytes with inflammatory infiltrate in the centrilobular
region in three of four mice. At 14 and 35 days, liver sections of
tPA+/uPA+ mice and
tPAo littermates were normal and
indistinguishable. Normalization of centrilobular injury was delayed in
uPAo mice, with near resolution seen 14 to 35
days after the initial challenge (Figure 3)
. These data suggest that in
the absence of tPA, uPA can independently promote the reparative
response after CCl4 injury and nearly normalize
hepatic histology within 7 days. In contrast, tPA cannot support
efficient repair in the absence of uPA, as demonstrated by a persistent
centrilobular injury at 14 days in uPAo livers.
Nevertheless, tPA remains an important physiological adjunct in view of
the profound delay in normalization of lobular appearance of livers
lacking both tPA and uPA; these livers display persistent centrilobular
injury as far out as 35 days after a single dose of
CCl4 (Figure 3)
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Plasminogen activators have been previously linked to
hepatocyte proliferation,20-25
and one potential
mechanism underlying the defective repair of
tPAo/uPAo livers is
impaired proliferative response. Using BrdU incorporation by
hepatocytes as an indicator of proliferation, we found that mice
lacking plasminogen activators mounted a proliferative response that
was similar to control animals in regards to localization of
BrdU-labeled cells within the hepatic lobule and degree of
proliferation (Figure 4)
. BrdU-labeled
hepatocytes were uniformly dispersed along noninjured areas of the
liver lobule in an indistinguishable manner in both targeted and
nontargeted littermates (Figure 4A)
. Analysis of the number of
BrdU-labeled hepatocytes 2 days after CCl4 showed
a >10-fold increase greater than baseline levels in all livers
regardless of the genotype. Although the increase tended to be more
pronounced in the livers of
tPAo/uPAo mice, this
finding did not reach statistical significance (Figure 4B)
.
Interestingly, the number of BrdU-labeled hepatocytes returned to near
baseline levels in nontargeted littermates as well as in
tPAo/uPAo mice despite the
persistence of pronounced centrilobular tissue damage at 7 and 14 days.
This apparent dissociation between proliferative response and hepatic
repair probably results from the restoration of normal cellular mass in
mice lacking plasminogen activators leading to normalization of hepatic
function. The maintenance of plasma albumin at normal levels at both
time points in control and plasminogen activator-deficient mice is
consistent with a prompt restoration of liver function (data not
shown). Interestingly, the coexistence of an adequate proliferative
response and the inability to clear necrotic cells in
uPAo and
tPAo/uPAo livers resulted
in an increase in liver mass in these mice to 47 to 49% greater than
control and tPAo littermates at 14 days (Figure 5)
.
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Increased uPA activity and plasminogen activation in
liver remnants after partial hepatectomy have been suggested to play a
regulatory role in liver regeneration.20,22
Therefore, we
used zymography to define the hepatic activity of plasminogen
activators after a toxic injury and to explore whether the loss of an
individual plasminogen activator species is accompanied by a
compensatory increase in the level of the remaining plasminogen
activator in injured livers. In
tPA+/uPA+ mice, liver
injury resulted in a rapid and substantial increase in the activity of
hepatic plasminogen activators, primarily uPA (Figure 6)
. The activity of plasminogen
activators progressively returned to basal levels throughout the 14-day
study period, seemingly in parallel with the microscopic resolution of
centrilobular damage. A similar pattern of transiently increased
activity of hepatic uPA was seen in tPAo mice
after CCl4 administration, although the decrease
in uPA activity appeared delayed. Interestingly, the transient change
in hepatic uPA activity coincided with the timely resolution of the
centrilobular injury of tPAo livers, suggesting
that uPA-mediated proteolysis efficiently directs hepatic repair. In
uPAo mice, hepatic tPA activity was markedly
increased at 7 and 14 days; however, based on the slow normalization of
hepatic architecture in these mice, the elevated tPA activity was not
sufficient to direct timely liver repair. Taken together, these data
point to a coordinate regulation of plasminogen activator expression
within an injured liver tissue that serves to promote repair. When this
combined proteolytic response is completely abolished by the
simultaneous inactivation of the tPA and uPA genes the reparative
response to the injury is profoundly impaired.
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At 7 and 14 days after injury, the most prominent histological
feature of tPAo/uPAo mice
was the presence of necrotic hepatocytes and accumulation of
eosinophilic material in the centrilobular region. This finding
suggested that the absence of plasminogen activators resulted in
defective proteolysis of cellular debris and/or extracellular matrix
that occurs during normal liver repair. Because fibrin and fibronectin
are important components of the provisional matrix within hepatic and
extra-hepatic wound fields,15,32
we explored whether
impaired clearance of these matrix elements is associated with the
defect in liver repair observed in
tPAo/uPAo mice.
Immunohistochemical analysis of liver sections 2 days after
CCl4 showed that fibrin (Figure 7)
and fibronectin (not shown) deposition
was a prominent feature of damaged centrilobular zones of all mice,
regardless of the genotype. In livers of
tPA+/uPA+ mice, clearance
of both substrates occurred concomitantly with resolution of the
lobular injury, which is consistent with the effective proteolytic
clearance of provisional matrix during repair. In contrast, impaired
fibrin/fibronectin clearance was noted in all areas of defective repair
in livers of mice lacking plasminogen activators. In
tPAo mice, there was minimal residual
fibrin/fibronectin in centrilobular areas beyond day 7, suggesting that
induction of proteolysis by its functional counterpart, uPA, can
efficiently direct clearance of both substrates in the liver. The
primary importance of uPA in fibrin/fibronectin clearance is further
supported by the persistent accumulation of fibrin/fibronectin in
diseased centrilobular areas 7 to 14 days after
CCl4 in uPAo livers.
However, the even more severe impairment in fibrin/fibronectin
clearance observed in
tPAo/uPAo livers at both 7
and 14 days clearly points to a strong functional cooperation between
both plasminogen activators in clearance of fibrin-related matrices
during liver repair.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reparative response of the liver after hepatocellular necrosis requires proliferation of relatively sedentary hepatocytes, matrix synthesis, and reorganization of the lobule. In support for the role of uPA as a facilitator of cellular proliferation during liver regeneration,20-25 we found a robust induction of hepatic uPA activity in nontargeted mice after CCl4, which correlates with a previous report of increased uPA activity in the regenerating rat liver.22 Despite these data, deficiency of either or both plasminogen activators in vivo seems to have no untoward effect on hepatocyte proliferation after an acute toxic injury. The number of hepatocytes entering the cell cycle and the distribution of proliferating cells along the noninjured portion of the liver lobule were unaffected by the absence of uPA and/or tPA. However, these findings do not conflict with or argue against the reported mitogenic role for uPA during liver regeneration.24 Rather, our results highlight important differences among the molecular pathways regulating the hepatic response to a physical and toxic injury.33-35 The finding of normal hepatocyte proliferation in tPAo/uPAo mice after a toxic injury suggests the existence of tPA/uPA-independent molecular pathways that effectively direct the restoration of liver mass. Nevertheless, despite the appropriate proliferative response, the ability of hepatocytes to populate diseased centrilobular areas is severely impaired because of a defect in the removal of components of provisional matrix and clearance of necrotic cells.
The striking similarities between the reparative defects observed in our studies of tPAo/uPAo mice and previous studies in plasminogen-deficient mice10 strongly imply that the failure to restore normal liver architecture is mechanistically because of a failure in local plasmin-mediated proteolysis, rather than some independent functional property of plasminogen activator (eg, uPAR-mediated signaling events)36-39 or plasminogen (eg, angiostatin-mediated events).40 Two critical questions that remain to be resolved are: 1) What target(s) of plasmin-mediated proteolysis is critical to the clearance of necrotic debris and reorganization of damaged tissue? and 2) Is there a general role of plasmin in re-organizing necrotic tissue in organ systems other than the liver? Interestingly, loss of plasmin-mediated proteolysis is known to be a major impediment to repair of other tissues (eg, skin incisional wounds15 and corneal scrape wounds41,42 ), and these defects can be effectively corrected by the genetic elimination of fibrin(ogen).16 Therefore, fibrin must be considered as a prime suspect in the search for plasmin targets related to hepatic repair after toxin injury. Indeed, fibrin is a persistent feature in the damaged centrilobular zones in tPAo/uPAo mice, and an inability to infiltrate, organize, and clear fibrin may be sufficient to impede hepatic repair. Nevertheless, it is highly unlikely that fibrin is the sole target of the plasminogen activation system that is relevant to hepatic repair. Notably, unlike repair in the skin, the absence of fibrin(ogen) does not restore normal liver repair in plasminogen-deficient mice after toxic injury.10 Therefore, our current working hypothesis is that a failure of plasmin-mediated proteolysis of nonfibrin substrates (eg, necrotic cells and associated extracellular matrices) may impede repair as effectively as a failure of plasmin-mediated fibrin clearance.
Proteolysis within the field of injury is critical to tissue repair through removal of matrix components and clearance of necrotic cells. In wound environments where tissue necrosis is limited (eg, skin incisional wounds), the most important physiological target of plasmin may be fibrin-rich matrices.16 In contrast, when cellular necrosis is the main feature of an injured tissue (eg, liver), fibrin-unrelated matrix proteolysis and removal of necrotic cells must occur to ensure effective repair.10 Based on the findings in the liver, one might anticipate that the absence of circulating fibrinogen is unlikely to restore the normal reparative pattern of cardiac26,43,44 and hepatic tissues in mice with defects in the plasminogen activation system. Together, these data point to a potential defect in nonfibrin targets that may relate to tissue repair, perhaps through plasmin-mediated activation of transforming growth factor or metalloproteinases.26,43,44 Alternately, effective removal of necrotic cells may be hampered by a defect in recruitment and/or activation of inflammatory cells within the field of injury. In this regard, using the same model of acute CCl4 injury in plasminogen-deficient mice, we found no obvious impairment in recruitment of inflammatory cells to damaged centrilobular regions after the toxic injury; however, mononuclear phagocytes failed to ingest abundant cellular debris from neighboring necrotic hepatocytes or within distorted sinusoidal spaces (Jorge A. Bezerra, unpublished work). Therefore, the plasminogen system may be critical to mononuclear phagocyte-mediated proteolytic clearance of necrotic cells during tissue repair.
In conclusion, these studies reveal a key regulatory role of tPA and uPA in the reparative response of the liver to an acute injury. The shared features of defective repair in mice lacking plasminogen activators and plasminogen strongly imply that plasmin serves a central proteolytic role in tissue repair. Future studies will determine whether tPA and uPA act solely by generation of plasmin-mediated proteolysis, or whether one or both proteases also act independently of plasmin generation to support tissue repair. Finally, it remains to be established what nonfibrin targets of the plasminogen activation system are important in the reorganization of necrotic tissue.
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Acknowledgements |
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Footnotes |
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Supported in part by the Hoffman-LaRoche, Ltd., Grant ROTRF 477924785 (to J. A. B.) and National Institutes of Health grants DK 55710 (to J. A. B.) and HL 47826 (to J. L. D.).
Accepted for publication November 17, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth factor-ß1 in acute rat liver injury. Growth Factors 1989, 1:219-226[Medline]
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