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Regular Articles |
From the Joseph J. Jacobs Center for Thrombosis and Vascular
Biology and the Department of Molecular Cardiology*
and the Department of Anatomic Pathology,
Cleveland Clinic Foundation, Cleveland, Ohio; the Department of
Internal Medicine,
Division of Pulmonary and
Critical Care Medicine, University of Michigan Medical Center, Ann
Arbor, Michigan; and the W. M. Keck Center for Transgene Research
and the Department of Chemistry and
Biochemistry,§
University of Notre Dame,
Notre Dame, Indiana
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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 primary physiological fibrinolytic system is the plasminogen
activation pathway.10
Components of this system include
the zymogen, plasminogen (Pg); plasminogen activators, urokinase
(u-PA), and tissue plasminogen activator (t-PA); plasminogen activator
inhibitors, PAI-1 and PAI-2; plasmin (Pm), the enzymatically active
form of Pg responsible for the degradation of fibrin; and the Pm
inhibitor, 
2 anti-plasmin. The development of
mice deficient for components of the fibrinolytic system has provided
the means to examine the role of these components in various
physiological processes. These include hemostasis and cell migratory
events associated with inflammation, reproduction, angiogenesis, and
tumor metastasis.11-19
Indeed, studies in these mice have
confirmed the importance of Pg in fibrinolysis and maintenance of
vascular patency.20
Other indirect studies have
shown that Pm-generated fibrin(ogen) degradation products can serve as
chemoattractants for leukocytes, hence magnifying the inflammatory
response.21,22
Pm itself has been shown to act as a
chemoattractant agent specifically for macrophages.23
In
addition, Pm is thought to activate matrix
metalloproteinases,24
which are capable of degrading many
extracellular matrix components including collagen, elastin,
fibronectin, proteoglycan, and laminin,25
and hence can
indirectly facilitate cell migration. Therefore, Pm could be involved
directly or indirectly with several aspects of repair mechanisms after
acute lung injury.
It has been reported that overexpression of PAI-1 results in increased deposition of collagen and fibrin in bleomycin-treated mice.26 On the other hand, deficiency of PAI-1 attenuated the development of pulmonary fibrosis. In the former case, inhibition of Pg activation led to an increase in fibrin clots in the interstitium and alveolar spaces.26 Additionally, u-PA treatment has been shown to diminish the development of bleomycin-induced pulmonary fibrosis in rats27 and mice.28 This study showed decreased lung collagen levels after treatment with u-PA, demonstrating the potential use of Pg activators as therapeutic agents for pulmonary fibrotic disorders. The current investigation was designed to evaluate the mechanism by which Pg and its activators, u-PA and t-PA, are involved in the development of pulmonary fibrosis, using a model of inflammatory injury and fibrosis. Lack of one or more of the components of the fibrinolytic system resulted in alterations in the response to acute lung injury and development of pulmonary fibrosis. Based on these findings, the current investigation is aimed at further delineation of the roles of genes involved in the fibrinolytic system in repair of lung injury induced by bleomycin, which may serve as a model of oxidative lung injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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WT, Pg-/-, u-PA-/-, u-PAR-/-, t-PA-/-, of mixed (50:50) C57BL6/129 strain background were developed and characterized as previously described.11,12,19,29,30 Mice were housed in micro-isolation cages on a constant 12-hour light/12-hour dark cycle in a temperature- (22.2°C) and humidity-controlled room and given ad libitum access to food and water. Sexually mature Pg+/- females were mated with Pg+/- males, and t-PA-/-, u-PA-/-, u-PAR-/- females were mated with their male counterparts. Experimental mice were 8 to 12 weeks of age and included males and females. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee.
Bleomycin Treatment
Intratracheal administration of a single dose of 0.075 U bleomycin (Bristol-Myers Squibb Co., Princeton, NJ) in 50 µl of sterile saline was performed by infusion through the vocal cords using a fiber optic light source for illuminating the entrance into the trachea while the mice were under intraperitoneal ketamine/xylazine anesthesia. This dose of bleomycin has been previously shown to produce pulmonary fibrosis in mice of similar genetic background.26 Control animals included nontreated and saline-treated mice. At defined time points after bleomycin infusion, mice were anesthetized with isofluorane and lung vasculature perfused free of blood by slowly injecting 3 ml of phosphate buffered saline (PBS) into the right ventricle.
Hydroxyproline Assay
To estimate the total amount of collagen in the lung,
hydroxyproline was measured as described26
from bleomycin-
or saline-treated mice 14 days after treatment. The lung vasculature
was perfused as described and the left lung was excised and homogenized
in 2 ml cold PBS using a hand homogenizer. A 1-ml aliquot of the
homogenate was vacuum-dried, weighed, and hydrolyzed in 6 N HCl at
110°C for 12 hours. Samples were filtered through a 45-µm filter
and 50 µl of the filtered sample was used for the assay which was
conducted in duplicate. An aliquot of 50 µl of citrate/acetate buffer
(5% citric acid, 1.2% glacial acetic acid, 7.24% sodium acetate,
3.4% sodium hydroxide) and 1 ml chloramine T solution (0.564 g
chloramine T, 4 ml n-propanol, 32 ml citrate/acetate buffer) were added
to the sample and allowed to incubate at room temperature for 20
minutes. A 1-ml aliquot of Ehrlich’s solution (4.5 g
-dimethylaminobenzaldehyde in 18.6 ml n-propanol, 7.8 ml of 70%
perchloric acid) was added to the sample and incubated at 65°C for 15
minutes. Absorbance was measured at 550 nm and the amount of
hydroxyproline was determined against a standard curve generated from
known concentrations of reagent hydroxyproline (Sigma Chemical Co., St.
Louis, MO). Samples containing known amounts of purified collagen
(Vitrogen-100, Celtrix Laboratories, Palo Alto, CA) were used to
confirm the ability of the assay to completely hydrolyze and recover
hydroxyproline from collagen.
Isolation and Identification of Lung Leukocytes
To study the inflammatory response during the acute phase after lung injury, leukocytes were isolated from whole lungs.31 Mice were anesthetized at day 0 and at 3, 5, 7, or 9 days after treatment with bleomycin or vehicle and lung vasculature was perfused free of blood. The left lung was excised and the trachea and hilar nodes were removed from the lung sample. Lungs were minced and digested in 15 ml of a digest solution containing collagenase (Type A, 1 mg/ml, Sigma Chemical); DNase I (50 U/ml, Sigma Chemical); Hanks’ balanced salt solution (HBSS; Gibco BRL, Grand Island, NY); antibiotics (Pen-Strep, 1%, Gibco BRL); and supplemented with 5% fetal calf serum (Gibco BRL). After digestion, the tissue was mechanically disrupted by multiple passages through a 10-ml plastic syringe. Contaminating erythrocytes were lysed in 0.075% NH4Cl in Tris-HCl, pH 7.2, and the leukocytes recovered free of cell debris by filtration through a nytex screen (3–100/35 mesh; Tekco, Briarcliff Manor, NY). To ensure a homogeneous leukocyte preparation, a 40% Percoll (Sigma Chemical) gradient was used and the leukocyte pellet resuspended in HBSS media containing antibiotics and serum. Cell viability was determined using trypan blue exclusion and total number of leukocytes per lung were quantitated using a hemocytometer.
To determine the type of leukocytes obtained from the lung digests, 100-µl samples containing 106 cells/ml were centrifuged onto a glass slide using a cytocentrifuge (Shandon, Sewickley, PA). Samples were then stained with Wright stain (EM Science, Gibbstown, NJ) and visualized by light microscopy. The number of macrophages was determined for a particular field of view (200 cells) in duplicate and expressed as mean ± SE.
Western Blot Analysis
To determine the protein expression and activation of MMP-12 whole lung extracts from saline- or bleomycin-treated WT and Pg-/- mice were homogenized in 2 ml of PBS containing 1% sodium dodecyl sulfate (SDS) and proteinase inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10-5 mol/L leupeptin, 1 mmol/L EDTA). Protein was extracted for 1 hour at room temperature, centrifuged, and the supernatant was used for total protein determination using the bicinchoninate (BCA, Pierce, Rockford, IL) method. SDS-polyacrylamide gel electrophoresis (PAGE) was performed using 80 µg total protein. Proteins were transferred onto an immobilon-P membrane (Millipore, Bedford, MA) and nonspecific protein binding was blocked in 5% powdered milk Tris-buffered saline (TBS) containing 0.05% Tween-20 (BLOTTO). A polyclonal antibody against recombinant mouse MMP-12 was kindly provided by Dr. Steve Shapiro (Washington University School of Medicine, St. Louis, MO). Membranes were incubated in anti-MMP-12 antibody (1:2000 in BLOTTO) at 4°C overnight. Membranes were washed in TBS-Tween, reblocked, then incubated with HRP-conjugated swine anti-rabbit IgG antibody (1:2000 in BLOTTO) and washed again. Finally, membranes were developed using the ECL Plus Kit (Amersham, Arlington Heights, IL).
Histology and Immunohistochemistry
To study the histological changes and alterations in the deposition of fibrin and collagen in situ during the acute and organizing stages after lung injury, right lungs from bleomycin- and saline-treated mice were examined at 7 and 14 days after treatment. Animals were anesthetized and perfused as described above. After excision of the left lung for other studies, such as collagen content, the right lung was instilled intrabronchially with Histochoice (Amresco, Solon, OH). The airway was then ligated, excised, and embedded in paraffin. Paraffin sections 7 µm thick were placed on slides coated with polyL-lysine (Sigma Chemical). Tissue sections were deparaffinized, rehydrated, and immersed in running water (for hematoxylin and eosin, Masson’s trichrome, and Prussian blue stains) or TBS/bovine serum albumin (BSA) or PBS/BSA (for immunohistochemistry).
Fibrin was identified by immunohistochemistry using a polyclonal antibody against mouse fibrin(ogen) (DAKO Corp., Carpinteria, CA). Histological sections were incubated with peroxidase-labeled rabbit anti-goat IgG (Dakopatts, Copenhagen, Denmark) and peroxidase activity was developed by incubation in 0.05 mol/L Tris-HCl, pH 7.0, containing 0.06% 3,3-diaminobenzidine and 0.1% H2O2. MMP-12 was localized in sections of lung using the same anti-MMP-12 antibody used for Western blots at 1:500 in PBS containing 0.1% BSA. Sections were incubated with biotinylated secondary antibody, followed by the ABC reagent from Vectastain ABC Kit (Vector Laboratories, Burlingame, CA). Peroxidase activity was detected as described above. Counterstaining was performed with hematoxylin.
Statistical Analysis
Values were expressed as means ± SE. Comparisons were made using Student’s t-test and analysis of variance when more than two values were compared. P values <0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because collagen content has been used as a measure of the degree
of fibrosis in the lung,32
these measurements were made in
the lungs of saline- or bleomycin-treated mice. Eitzman and
others26
have demonstrated that pulmonary fibrosis peaks
14 days after bleomycin treatment in mice of a similar genetic strain.
Therefore, lungs were examined 14 days after treatment except for
t-PA-/- mice who survived
only 11 days post-drug treatment. After intratracheal administration of
bleomycin, collagen levels increased significantly above saline
controls in Pg-/-,
Pg+/-, and t-PA+/- mice
(150.06 ± 28.40 vs. 33.53 ± 6.00,
P = 0.02; 147.02 ± 25.70 vs.
60.07 ± 1.6, P = 0.04; 152.70 ± 8.50
vs. 55.67 ± 8.20, P = 0.0005 µg
hydroxyproline/left lung; Figure 1
). In
WT, u-PA-/-, and
u-PAR-/- mice there was
also an increase in collagen with values approaching significance
(89.38 ± 19.20 vs. 47.97 ± 25.80,
P = 0.24; 100.90 ± 15.14 vs.
63.53 ± 5.30, P = 0.12; 78.76 ± 11.60
vs. 50.70 ± 7.45, P = 0.18 µg
hydroxyproline/left lung; Figure 1
). Collagen levels observed in
Pg-/- and
t-PA-/- mice were higher
than those observed in WT,
u-PA-/-, and
u-PAR-/- mice
(150.06 ± 28.4 and 152.70 ± 8.50 µg hydroxyproline/left
lung, respectively, vs. 89.38 ± 19.20 µg
hydroxyproline/left lung for WT mice; Figure 1
).
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To better understand the histological changes associated with this
acute lung injury model, the right lungs from bleomycin- or
saline-treated mice were collected 14 days after treatment. Lung tissue
sections were stained with hematoxylin and eosin, Masson’s trichrome
(for collagen identification), or Prussian blue (for identifying areas
of chronic hemorrhage). The lungs of saline-treated mice appeared
normal regardless of genotype or time point after treatment. Collagen
was identified in these lungs only in areas surrounding large vessels
and airways (Figure 2A)
. At 14 days after
bleomycin treatment, focal areas of fibrosis in the interstitium as
well as proteinaceous exudate in the intra-alveolar spaces were
observed in some areas of the lungs of WT mice (Figure 2B)
. However, an
unexpected hemorrhage phenotype was observed in the lungs of these mice
at this time point (Figure 2C)
. Associated with these areas of
hemorrhage were hemosiderin-laden macrophages, as observed in adjacent
tissue sections stained with Prussian blue, indicating chronic
hemorrhage (Figure 2D)
. In contrast, all bleomycin-treated
Pg-/- mice examined
(n = 7) showed extensive areas of fibrosis but
no areas of hemorrhage (Figure 2E)
. In these animals, abnormal collagen
deposition was observed in the interstitium associated with the areas
of fibrosis (Figure 2F)
. Pg+/- mice showed a
mixed phenotype of hemorrhage, observed in the WT mice, and collagen
content and fibrotic lesions, found in the
Pg-/- mice (Figure 2G)
.
Chronic hemorrhage observed in these mice was confirmed by Prussian
blue staining of adjacent sections, which showed the presence of
hemosiderin-laden macrophages in areas of hemorrhage (Figure 2H)
.
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. Similar to
Pg-/- mice, no hemorrhage
was found in any of these animals.
u-PAR-/- mice exhibited
focal areas of fibrosis as observed in the WT mice (Figure 3B)
. Similar
to Pg-/- mice, none of
these animals showed signs of chronic intra-alveolar hemorrhage as
confirmed by the absence of positive cells in adjacent sections stained
with Prussian blue (not shown). In contrast,
t-PA-/- mice presented a
mixed phenotype of hemorrhage found in the WT and
Pg-/- animals and
extensive fibrosis found in
Pg-/- and
u-PA-/- mice (Figure 3C)
.
The fibrotic lesions in all of the animals studied showed the typical
thickening of the alveolar wall and the presence of abnormal deposition
of collagen in the interstitium. The differences in the extent of
abnormal collagen deposition observed in the different genotypes
correlated with the accumulation of collagen in these mice as measured
by hydroxyproline levels (Figure 1)
. Interestingly, the hemorrhage
phenotype observed in the WT,
Pg-/-, and
t-PA-/- mice correlated
with the early deaths observed in these animals, Table 1
. Moreover, earlier death exhibited in
the Pg+/- and
t-PA-/- mice could also
be due to their enhanced accumulation of fibrotic lesions compared to
the WT mice. In that regard, early deaths of
u-PA-/- mice could be the
result of the extensive fibrosis observed in these animals as early as
day 7 after bleomycin treatment. However, no early deaths were found in
Pg-/- or
u-PAR-/- mice.
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Studies using thioglycollate14 as a model of inflammation have shown quantitative differences between WT and Pg-/- mice in macrophage levels in the peritoneum. Therefore, macrophage levels during the acute stage after bleomycin treatment of Pg-/-, u-PA-/-, u-PAR-/-, t-PA-/-, and WT mice were studied. Leukocytes were isolated from collagenase-digested whole lungs at days 0 (no treatment, baseline), 3, 5, 7, and 9 after saline or bleomycin treatment. This method yielded samples containing >95% leukocytes as confirmed by positive CD45 immunostaining (data not shown). The leukocytes isolated in this way represented those present in the alveolar spaces as well as in the interstitium of the lung at the time of collection.
No significant increase was observed in the number of macrophages
present in the lungs of mice at 3 days after bleomycin treatment,
although WT mice showed the highest levels of all of the genotypes
studied (Table 2)
. Macrophage levels at
this time point were comparable to those found in the saline-treated
counterparts of Pg-/-,
u-PA-/-, and
t-PA-/- mice, and may
represent the alveolar macrophages already present in the lung (Table 2)
. The peak in the macrophage levels in the lungs of WT mice occurred
5 days after bleomycin treatment (Table 2)
. This was also the peak time
for the accumulation of macrophages in the lungs of
Pg-/- (4.86 ±
0.40 x 106) and
u-PA-/- (5.62 ±
0.70 x 106) mice treated with bleomycin.
However, their levels were considerably less than those found in WT
mice (8.68 ± 0.60 x 106),
P = 0.001 relative to
Pg-/- and
P = 0.04 relative to
u-PA-/- (Table 2)
. These
findings are consistent with those observed in
Pg-/- mice using
thioglycollate- and biopolymer-induced peritoneal inflammation
models.14
In the present study, at day 7 post-treatment,
there was a decrease in the number and percentage of macrophages found
in the lungs of Pg-/-,
u-PA-/-, and WT mice
(Table 2)
. The decrease in macrophage counts at this time point
coincided with an increase in the percentage of lymphocytes found in
the lungs of bleomycin-treated mice (data not shown). This was not the
case for t-PA-/- mice
treated with bleomycin, in which macrophage numbers continued to
increase reaching levels comparable to those of WT mice at 9 days after
bleomycin treatment (Table 2)
. No increase in the macrophage levels in
the lungs of u-PAR-/-
mice treated with bleomycin was observed between 3 and 7 days after
bleomycin treatment. However, a significant increase in macrophage
number was observed at day 9 after bleomycin treatment compared to
their saline controls (6.22 ± 0.50 vs. 2.73 ±
0.70, P = 0.05) although at this late time point the
level did not reach the highest observed level in WT mice (Table 2)
.
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Because one of the first events after acute lung injury is the
extravasation of plasma proteins such as fibrinogen,32
accumulation of fibrin in the intra-alveolar spaces and interstitium of
the lung of bleomycin- or saline-treated mice was analyzed. Therefore,
the extent and localization of fibrin(ogen) in the lungs of these mice
were studied at 3, 7, and 14 days after treatment. No fibrin(ogen)
deposits were observed in the lungs of WT animals treated with saline
at any time point studied (data not shown). In contrast, as described
earlier,11,12
Pg-/- control mice showed
spontaneous fibrin deposition in the lungs throughout the time points
studied. Bleomycin-treated lungs of WT mice showed few areas of
fibrin(ogen) deposits at 3 and 7 days after bleomycin treatment, which,
for the most part, were resolved by day 14 (Figure 4A)
. In sharp contrast,
Pg-/- mice showed
extensive fibrin (ogen) deposition as early as day 3, peaking at day 7,
and continuing even at day 14 after bleomycin treatment (Figure 4, B and C)
. The areas of fibrin(ogen) deposition in the
Pg-/- mice were
associated with regions of fibrosis (Figure 4C)
.
u-PA-/- mice also showed
extensive areas of fibrin(ogen) deposition in the lung interstitium
which were associated with areas of fibrosis (data not shown).
Additionally, t-PA-/-
mice also exhibited areas of fibrin(ogen) deposits, especially in the
vasculature of the lung, where fibrin thrombi could be observed (data
not shown).
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Because Pm is a known activator of
pro-MMPs24,33
the expression, localization, and
activation of MMPs in whole lung extracts of saline- or
bleomycin-treated mice were analyzed. Of particular interest was
MMP-12, also known as macrophage metalloelastase.34
The
pro-enzyme form of MMP-12 (54 kd) was present in whole lung extracts
from both WT and Pg-/-
mice as early as 5 days after treatment with bleomycin (Figure 5)
. However, the active, 22-kd form of
the enzyme was observed only in WT animals at this time point (Figure 5)
. Neither the proenzyme nor the active form was observed in either
genotype by day 14 after bleomycin treatment (Figure 5)
.
Immunohistochemical studies using the same antibody to localize MMP-12
showed that the enzyme is present in the alveolar wall of WT mice
associated with areas of hemorrhage in these animals (Figure 6A)
. Specifically, MMP-12 seemed to be
localized in the capillary component of the alveolar wall (Figure 6B)
.
The specific MMP-12-positive cell type in the capillary of the alveolar
wall remains to be determined. However, macrophages are potential
candidates, because they are the only cell type known to produce this
enzyme.34
In contrast, MMP-12 was not present in areas of
fibrosis in the WT or
Pg-/- mice (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several animal models have been developed to study the mechanisms of lung repair after acute and chronic inflammatory injury. Cytotoxic drugs are the most widely used to induce pulmonary fibrosis and among these the antibiotic bleomycin has been the most extensively studied.39-41 Intratracheal administration of bleomycin in mice and rats causes both the inflammatory and fibrotic responses observed in patients with pulmonary fibrosis.42 In this study, bleomycin was used to study the role of the fibrinolytic system in lung repair after injury and the development of pulmonary fibrosis.
The first phase of the lung response to injury is characterized by severe inflammation. Initial recruitment of inflammatory cells into the alveolar spaces is brought about by chemoattractant agents derived from the injured lung tissue. Injury causes increased permeability of pulmonary epithelium and endothelium, resulting in extravasation of plasma proteins and, ultimately, formation of fibrin clots in the alveolar lumen.35 Pertinent to this study, Pm-catalyzed fibrinolysis generates a series of high molecular weight fibrin degradation products (FDPs) that can serve as chemoattractants to leukocytes.21,22 Therefore, the inflammatory response could be augmented with the recruitment of new inflammatory cells by these potent FDPs. Peak levels of macrophages occurred at day 5 for WT, u-PA-/-, and Pg-/- mice, although levels were significantly diminished in u-PA-/- and Pg-/- mice relative to WT mice. A delayed, but significant, increase in macrophage levels was observed in t-PA-/- mice, whereas the delayed response in u-PAR-/- mice never reached the peak levels observed in t-PA-/- and WT mice. The low macrophage levels in u-PAR-/- mice during the early stages of repair may be due to a necessity for an intact uPA/uPAR system for macrophage motility.43 On the other hand, low macrophage levels in u-PAR-/- mice could be related to a u-PA independent function for this protein. It has been demonstrated that a lack of u-PAR results in compromised ß2-integrin activation, which has a deleterious effect on leukocyte-endothelial cell adhesion and resultant transendothelial migration.44 Additionally, ICAM-1, a ligand for ß2-integrins, has been identified in bleomycin-injured lungs in rats,45 and, therefore, u-PAR/ß2-integrin interactions may play a significant role in inflammatory cell migration in response to bleomycin-induced lung injury. The eventual increase in macrophage levels in u-PAR-/- mice may be the result of activation of other adhesion receptors independent of the u-PAR/ß2-integrin system during the later stages of the inflammatory response. These observations implicate u-PA-mediated activation of plasminogen in contributing to the enhanced influx of macrophages into the lung after acute injury.
In the current studies, areas of alveolar hemorrhage were observed in
WT and t-PA-/- mice but
not in u-PA-/-,
u-PAR-/-, and
Pg-/- mice. The
hemorrhagic areas were characterized by the presence of
hemosiderin-laden macrophages, indicative of chronic hemorrhage in the
lungs of these mice. Similar phenotypes have been observed in studies
of myocardial infarction with mice deficient for components of the
fibrinolytic system.16
In these latter studies,
Pg-/- and
u-PA-/- mice did not show
the ventricular hemorrhage that was present in WT counterparts, as well
as in t-PA-/- mice. Taken
together, these investigations support the involvement of the u-PA/Pg
system in mediating the hemorrhagic phenotype observed in these two
animal models (Figure 7)
. This is
underscored by the clinical observation that intra-alveolar hemorrhage
has been shown to occur during the alveolar edema phase of patients
with acute respiratory distress syndrome.46
|
.
One of the prominent phenotypes in bleomycin-induced pulmonary fibrosis
is extensive abnormal collagen deposition in the interstitium. It was
previously shown that overexpression of PAI-1 resulted in increased
deposition of collagen in the lungs of bleomycin treated mice, whereas
PAI-1-/- mice had levels
comparable to those of PBS-treated control mice.26
This
suggests that Pm activity plays a key role in the regulation of
collagen accumulation in the lungs after acute lung injury.
Histologically, this study demonstrated enhanced fibrotic lesion
development in bleomycin-treated
Pg-/-,
Pg+/-,
t-PA-/-, and
u-PA-/- mice relative to
WT and u-PAR-/- mice.
Additionally, hydroxyproline levels, a measure of collagen content, in
the lungs of these mice were all enhanced at 14 days after drug
treatment. Pg-/- mice
demonstrated 1.7-fold levels of hydroxyproline over that observed in WT
mice. Plasminogen levels in the blood of Pg+/-
mice have been shown to be approximately one-half of that observed in
WT mice (35 ± 2 µg/ml vs. 84 ± 8
µg/ml),11
and even at those levels a dramatic effect on
collagen deposition was observed in Pg+/- mice
which was similar to that observed for
Pg-/- mice. The levels of
hydroxyproline found in the
u-PA-/- mice were similar
to those observed in WT animals at 14 days, even though the lungs
appeared more fibrotic on histological examination. These findings
contrast with those of Lardot and others52
using a
crystalline silica model of alveolitis that progressed to fibrosis in
u-PA-/- mice. In that
study an increase in hydroxyproline levels in the
u-PA-/- mice was found
compared to their WT counterparts. This correlated with the development
of fibrotic lesions in these mice 30 days after treatment. However, it
was also noted that the fibrinolytic response varied depending on the
agent used to promote alveolitis. This differential response may be
related to selective tissue responses to different agents and may
explain the discrepancy observed in hydroxyproline levels in
u-PA-/- mice from these
two studies. It is also well known that collagen deposition is a
balance between processes of synthesis and degradation that is
regulated by a complex network of cytokines, ie, transforming growth
factor-ß (TGF-ß), interleukin-1 (IL-1), and tumor necrosis
factor- (TNF-).32
In the case of
u-PA-/- mice, the absence
of u-PA could result in altered expression of important mediators of
collagen synthesis independent of plasmin activity. For example,
studies have indicated that the release of IL-1 by macrophages is
inhibited by PAI-I.53
IL-1, which is known to increase
fibroblast collagen synthesis, has been shown to be increased in
bleomycin-induced pulmonary fibrosis.54
Additionally,
studies have shown that u-PA amplifies TNF- secretion of THP-1
mononuclear phagocytes independent of plasmin activity.55
Therefore, u-PA could potentially play diverse roles in the repair
process after acute lung injury.
Bleomycin-induced lung injury has been shown to result in the
accumulation of fibrin in the alveolar and interstitial spaces of the
lung.56
These fibrin deposits colocalized with the
abnormal collagen deposited in the lung interstitium, which was
associated with fibroproliferative lesions. Additionally, fibrin has
been shown to serve as a scaffolding molecule for the migration and
proliferation of fibroblasts.57,58
Therefore, the
inability of Pg- and Pg activator-deficient mice to clear fibrin
deposited into the alveolar spaces and interstitium would potentially
result in the proliferation and migration of fibroblasts into the space
once occupied by the alveoli, leading to the development of pulmonary
fibrosis and end stage honeycomb appearance of the lung (Figure 7)
.
In conclusion, the studies reported here have shown that the repair process after acute oxidative lung injury depends on the balance between deposition and breakdown of matrix molecules such as fibrin and collagen. As a result, differences in the expression of components of the fibrinolytic system alter this balance by locally affecting fibrinolysis, the inflammatory response, and activation of matrix metalloproteinases at the site of lung injury.
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Acknowledgements |
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Footnotes |
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Supported by National Institutes of Health (National Heart, Lung and Blood Institute) RO1 grant HL63682–01 (to V. A. P.) and postdoctoral fellowship no. 9804562 from the American Heart Association, Midwest Affiliate (to C. M. S.).
Accepted for publication March 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Bß chains of fibrinogen stimulate proliferation of human fibroblasts. J Cell Sci 1993, 104:409-413[Abstract]
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 P. Shetty, T. Velusamy, Y. P. Bhandary, R. S. Shetty, M.-C. Liu, and S. Shetty Urokinase Expression by Tumor Suppressor Protein p53: A Novel Role in mRNA Turnover Am. J. Respir. Cell Mol. Biol., September 1, 2008; 39(3): 364 - 372. [Abstract] [Full Text] [PDF]
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 S. C. Bryer, G. Fantuzzi, N. Van Rooijen, and T. J. Koh Urokinase-Type Plasminogen Activator Plays Essential Roles in Macrophage Chemotaxis and Skeletal Muscle Regeneration J. Immunol., January 15, 2008; 180(2): 1179 - 1188. [Abstract] [Full Text] [PDF]
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 S. Shetty, T. Velusamy, S. Idell, P. Shetty, A. P. Mazar, Y. P. Bhandary, and R. S. Shetty Regulation of Urokinase Receptor Expression by p53: Novel Role in Stabilization of uPAR mRNA Mol. Cell. Biol., August 15, 2007; 27(16): 5607 - 5618. [Abstract] [Full Text] [PDF]
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 C. M. Swaisgood, M. A. Aronica, S. Swaidani, and E. F. Plow Plasminogen Is an Important Regulator in the Pathogenesis of a Murine Model of Asthma Am. J. Respir. Crit. Care Med., August 15, 2007; 176(4): 333 - 342. [Abstract] [Full Text] [PDF]
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 D. Cosgrove A complex, tissue-specific role for plasmin and its regulators in modulating fibrogenic activity Am J Physiol Renal Physiol, July 1, 2007; 293(1): F10 - F11. [Full Text] [PDF]
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S. Shetty, T. Velusamy, S. Idell, H. Tang, and P. K. Shetty Regulation of urokinase receptor expression by protein tyrosine phosphatases Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L414 - L421. [Abstract] [Full Text] [PDF]
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H. Matsuoka, T. H. Sisson, T. Nishiuma, and R. H. Simon Plasminogen-Mediated Activation and Release of Hepatocyte Growth Factor from Extracellular Matrix Am. J. Respir. Cell Mol. Biol., December 1, 2006; 35(6): 705 - 713. [Abstract] [Full Text] [PDF]
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 A. A. Eddy and A. B. Fogo Plasminogen Activator Inhibitor-1 in Chronic Kidney Disease: Evidence and Mechanisms of Action J. Am. Soc. Nephrol., November 1, 2006; 17(11): 2999 - 3012. [Full Text] [PDF]
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R. J. Tan, C. L. Fattman, L. M. Niehouse, J. M. Tobolewski, L. E. Hanford, Q. Li, F. A. Monzon, W. C. Parks, and T. D. Oury Matrix Metalloproteinases Promote Inflammation and Fibrosis in Asbestos-Induced Lung Injury in Mice Am. J. Respir. Cell Mol. Biol., September 1, 2006; 35(3): 289 - 297. [Abstract] [Full Text] [PDF]
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 H. Fujimoto, E. C. Gabazza, O. Taguchi, Y. Nishii, H. Nakahara, N. E. Bruno, C. N. D'Alessandro-Gabazza, M. Kasper, Y. Yano, M. Nagashima, et al. Thrombin-Activatable Fibrinolysis Inhibitor Deficiency Attenuates Bleomycin-Induced Lung Fibrosis Am. J. Pathol., April 1, 2006; 168(4): 1086 - 1096. [Abstract] [Full Text] [PDF]
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J. H. Kim, H. Y. Kim, S. Kim, J.-H. Chung, W. S. Park, and D. H. Chung Natural Killer T (NKT) Cells Attenuate Bleomycin-Induced Pulmonary Fibrosis by Producing Interferon-{gamma} Am. J. Pathol., November 1, 2005; 167(5): 1231 - 1241. [Abstract] [Full Text] [PDF]
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 Q. Cheng, Y. Zhao, W. E. Lawson, V. V. Polosukhin, J. E. Johnson, T. S. Blackwell, and D. Gailani The effects of intrinsic pathway protease deficiencies on plasminogen-deficient mice Blood, November 1, 2005; 106(9): 3055 - 3057. [Abstract] [Full Text] [PDF]
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A. de Giorgio-Miller, S. Bottoms, G. Laurent, P. Carmeliet, and S. Herrick Fibrin-Induced Skin Fibrosis in Mice Deficient in Tissue Plasminogen Activator Am. J. Pathol., September 1, 2005; 167(3): 721 - 732. [Abstract] [Full Text] [PDF]
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 A. D. Weisberg, F. Albornoz, J. P. Griffin, D. L. Crandall, H. Elokdah, A. B. Fogo, D. E. Vaughan, and N. J. Brown Pharmacological Inhibition and Genetic Deficiency of Plasminogen Activator Inhibitor-1 Attenuates Angiotensin II/Salt-Induced Aortic Remodeling Arterioscler. Thromb. Vasc. Biol., February 1, 2005; 25(2): 365 - 371. [Abstract] [Full Text] [PDF]
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M. H. Lazar, P. J. Christensen, M. Du, B. Yu, N. M. Subbotina, K. E. Hanson, J. M. Hansen, E. S. White, R. H. Simon, and T. H. Sisson Plasminogen Activator Inhibitor-1 Impairs Alveolar Epithelial Repair by Binding to Vitronectin Am. J. Respir. Cell Mol. Biol., December 1, 2004; 31(6): 672 - 678. [Abstract] [Full Text] [PDF]
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T. Nishiuma, T. H. Sisson, N. Subbotina, and R. H. Simon Localization of Plasminogen Activator Activity within Normal and Injured Lungs by In Situ Zymography Am. J. Respir. Cell Mol. Biol., November 1, 2004; 31(5): 552 - 558. [Abstract] [Full Text] [PDF]
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F. Buhling, C. Rocken, F. Brasch, R. Hartig, Y. Yasuda, P. Saftig, D. Bromme, and T. Welte Pivotal Role of Cathepsin K in Lung Fibrosis Am. J. Pathol., June 1, 2004; 164(6): 2203 - 2216. [Abstract] [Full Text] [PDF]
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N. Hattori, S. Mizuno, Y. Yoshida, K. Chin, M. Mishima, T. H. Sisson, R. H. Simon, T. Nakamura, and M. Miyake The Plasminogen Activation System Reduces Fibrosis in the Lung by a Hepatocyte Growth Factor-Dependent Mechanism Am. J. Pathol., March 1, 2004; 164(3): 1091 - 1098. [Abstract] [Full Text] [PDF]
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S. Chuang-Tsai, T. H. Sisson, N. Hattori, C. G. Tsai, N. M. Subbotina, K. E. Hanson, and R. H. Simon Reduction in Fibrotic Tissue Formation in Mice Genetically Deficient in Plasminogen Activator Inhibitor-1 Am. J. Pathol., August 1, 2003; 163(2): 445 - 452. [Abstract] [Full Text] [PDF]
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H. Fujimoto, E. C. Gabazza, O. Hataji, H. Yuda, C. N. D'Alessandro-Gabazza, M. Nakano, O. E. Franco, T. Hayashi, K. Suzuki, Y. Adachi, et al. Thrombin-activatable Fibrinolysis Inhibitor and Protein C Inhibitor in Interstitial Lung Disease Am. J. Respir. Crit. Care Med., June 15, 2003; 167(12): 1687 - 1694. [Abstract] [Full Text] [PDF]
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G. Zhang, H. Kim, X. Cai, J. M. Lopez-Guisa, P. Carmeliet, and A. A. Eddy Urokinase Receptor Modulates Cellular and Angiogenic Responses in Obstructive Nephropathy J. Am. Soc. Nephrol., May 1, 2003; 14(5): 1234 - 1253. [Abstract] [Full Text] [PDF]
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G. Zhang, H. Kim, X. Cai, J. M. Lopez-Guisa, C. E. Alpers, Y. Liu, P. Carmeliet, and A. A. Eddy Urokinase Receptor Deficiency Accelerates Renal Fibrosis in Obstructive Nephropathy J. Am. Soc. Nephrol., May 1, 2003; 14(5): 1254 - 1271. [Abstract] [Full Text] [PDF]
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C. Jakubzick, E. S. Choi, S. L. Kunkel, B. H. Joshi, R. K. Puri, and C. M. Hogaboam Impact of Interleukin-13 Responsiveness on the Synthetic and Proliferative Properties of Th1- and Th2-Type Pulmonary Granuloma Fibroblasts Am. J. Pathol., May 1, 2003; 162(5): 1475 - 1486. [Abstract] [Full Text] [PDF]
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T.-L. Tuan, H. Wu, E. Y. Huang, S. S. N. Chong, W. Laug, D. Messadi, P. Kelly, and A. Le Increased Plasminogen Activator Inhibitor-1 in Keloid Fibroblasts May Account for their Elevated Collagen Accumulation in Fibrin Gel Cultures Am. J. Pathol., May 1, 2003; 162(5): 1579 - 1589. [Abstract] [Full Text] [PDF]
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T. H. Sisson, K. E. Hanson, N. Subbotina, A. Patwardhan, N. Hattori, and R. H. Simon Inducible lung-specific urokinase expression reduces fibrosis and mortality after lung injury in mice Am J Physiol Lung Cell Mol Physiol, November 1, 2002; 283(5): L1023 - L1032. [Abstract] [Full Text] [PDF]
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V. A. Ploplis, J. Wilberding, L. McLennan, Z. Liang, I. Cornelissen, M. E. DeFord, E. D. Rosen, and F. J. Castellino A Total Fibrinogen Deficiency Is Compatible with the Development of Pulmonary Fibrosis in Mice Am. J. Pathol., September 1, 2000; 157(3): 703 - 708. [Abstract] [Full Text] [PDF]
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