|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
ABSTRACT |
|---|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
It is well recognized that activation of the coagulation system plays
an important role in bleomycin (BLM)-induced lung injury and fibrosis. The protein C (PC) pathway is an important regulator of
the coagulation system. In this study, we evaluated the bronchoalveolar lavage fluid (BALF) concentration of activated PC (APC) and
the therapeutic effect of the intratracheal administration of APC on BLM-induced lung fibrosis in mice. APC levels in BALF were significantly lower in BLM-treated animals than in the saline-treated group. Fibrotic changes were progressive in mice treated with BLM and intratracheal instillation of vehicle (BLM/Veh) after 14 and
21 d of BLM infusion. Compared with the BLM/Veh group, histologic findings on Days 14 and 21 in mice treated with BLM and
intratracheal instillation of APC (BLM/APC) showed less fibrotic lesions in the subpleural and central areas of the lung. The mean Aschcroft's fibrosis score in the BLM/Veh group was significantly (p < 0.05) higher than in the BLM/APC group. The lung hydroxyproline content on Day 21 was significantly higher (p < 0.05) in
the BLM/Veh group (1.78 ± 0.07 µmol/lung weight) than in the
BLM/APC (1.30 ± 0.06 µmol/lung weight) group. The ratio of plasminogen activator activity to thrombin level in BALF was significantly increased in the BLM/APC group compared with the BLM/
Veh group on Day 21. The expression of tumor necrosis factor-
and interleukin-1
was significantly decreased in the lungs of the
BLM/APC group compared with the BLM/Veh group on Day 14 after BLM infusion. These results showed that intratracheal APC
administration inhibits the development of lung fibrosis in BLM-induced lung injury, giving further support to the important role
that the PC pathway plays in the mechanism of lung fibrosis.
| |
INTRODUCTION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Lung injury induced by bleomycin (BLM) in animals is a well-characterized histologic and biochemical model of human pulmonary fibrosis. Animals develop pulmonary fibrosis when given BLM by intratracheal or intravenous administration or by over-1-wk continuous subcutaneous infusion (1). Administration of BLM results in a neutrophilic and lymphocytic acute pan-alveolitis in the first week (4). This acute inflammatory reaction is followed by a 2- to 3-wk subacute phase characterized by clearing of inflammatory cells, proliferation of fibroblasts, and synthesis of extracellular matrix proteins, eventually leading to perivascular, peribronchial, and subpleural fibrosis (4).
It is well recognized that the activation of the coagulation system and subsequent formation of a fibrin-rich intra-alveolar exudate play important roles in the lung tissue repair process after injury by BLM (5). In vitro experiments have shown that fibrin in combination with fibronectin serves as a part of a complex matrix upon which fibroblasts may proliferate and secrete connective tissue components leading to intra-alveolar and interstitial fibrosis (6). This fibrin matrix, its soluble degradation products, and locally generated thrombin may, in turn, modulate the tissue repair response by altering vascular permeability, by stimulating fibroblast and neutrophil migration, and by promoting adhesion and spreading of both endothelial cells and fibroblasts (7, 8). These ideas gain additional support for the demonstration of fibrin and markers of thrombin generation in the lung of patients with idiopathic pulmonary fibrosis (9).
The protein C (PC) pathway is an important regulator of
the coagulation system. The anticoagulant PC zymogen is converted to activated protein C (APC) by the thrombomodulin-
thrombin complex on the phospholipid surface of endothelial
cells, monocytes, and platelets (10). Classically, APC has been
described to exert anticoagulant activity by catalyzing the proteolytic inactivation of the coagulation factors, factor Va and
factor VIIIa, and profibrinolytic activity by inactivating plasminogen activator inhibitor type 1 (PAI-1) (11). The clinical
importance of the PC pathway for the regulation of the coagulation system is illustrated by the frequency of thromboembolic disease in subjects with heterologous or homologous deficiency of PC or protein S and the recently described resistance
to APC (12). In addition, recent in vivo studies have suggested that besides modulating the activation of blood coagulation, the PC pathway may also regulate the inflammatory response. Studies in experimental animals have shown that
APC may play a role in the inflammatory response by modulating the expression of cytokines such as tumor necrosis factor (TNF)- and by blocking neutrophil activation (15, 16).
These observations have been supported by more recent in vitro
studies in which it was shown that APC inhibits lipopolysaccharide-phorbol ester-induced and 
-interferon-induced production of proinflammatory cytokines, and suppresses E-selectin-mediated inflammatory cell adhesion to endothelial cells (16).
Further, the recent identification of the APC receptor suggests
that APC may directly exert anti-inflammatory activity by binding to its receptor on the cell surface (17, 18). In a previous
study, we reported that components of the PC pathway are
also detected in the bronchoalveolar lavage fluid (BALF) of
normal subjects and that activation of PC is decreased in
BALF of patients with interstitial lung disease and pulmonary fibrosis (19). Based on these previous observations, we hypothesized that APC may be of value for the treatment of lung
injury and pulmonary fibrosis. In the present investigation, we
evaluated therapeutic effect of intratracheal instillation of APC
on the BLM-induced pulmonary fibrosis in the mouse.
| |
METHODS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
Animals and BLM Administration
Pathogen-free, 8- to 10-wk-old, female, C57BL/6 mice, weighing 18 to 22 g, were purchased from Nihon SLC (Hamamatsu, Japan) and maintained in a specific pathogen-free environment in the animal house of our university. Lung damage was elicited by administering BLM (Nihon Kayaku, Tokyo, Japan) by constant subcutaneous infusion through osmotic minipumps (model 2001; Alza Corp., Palo Alto, CA), as originally described by Harrison and Lazo (2). BLM was dissolved in sterile saline and loaded into 7-d minipumps. For the control animals, the minipumps were loaded with sterile saline in a similar manner. Each animal was anesthetized with sodium pentobarbital at a dose of 62.5 mg/kg of mouse body weight given intraperitoneally. The minipump was implanted through a small incision in the mouse's back; the wound was then sealed with wound clips. After the mice were killed, the pumps were examined to determine if they had delivered the entire dosage of their contents in each mouse. All animal protocols were approved by Mie University's Committee on Animal Investigation.
APC Administration
The therapeutic effect of APC on BLM-induced lung fibrosis was evaluated by intratracheally administering to the animals APC dissolved in vehicle (20 mM Tris-HCl, 0.1 M NaCl, pH 7.4) or vehicle containing albumin. For APC administration, mice were anesthetized with pentobarbital as described previously. After exposing the trachea using sterile technique, either APC in vehicle or an equivalent volume (0.3 ml) of sterile vehicle containing albumin was slowly and directly instilled into the trachea lumen through a tube placed so as to allow homogenous distribution of APC in both lungs. There was no backward flow of liquid during the instillation of APC or vehicle into the trachea lumen. The skin wound was then closed and the animals were allowed to recover. The optimal day for the treatment of lung injury with APC was evaluated by treating mice with a single intratracheal instillation of APC (1 mg/kg of mouse body weight; Sigma, St. Louis, MO) or vehicle containing albumin (1 mg/kg of mouse body weight) on the third, seventh or fourteenth day of BLM infusion. BALF sampling from each group of animals (n = 4) was performed on Day 21 after BLM infusion. Samples from control animals receiving BLM pump infusion with intratracheal instillation of vehicle containing albumin (1 mg/kg of mouse body weight; BLM/Veh group) or saline pump infusion with intratracheal instillation of APC (1 mg/kg of mouse body weight; SAL/APC group) were also available for making comparison. For evaluating the dose-dependent effect of APC on BLM-induced lung injury, mice receiving BLM infusion were treated with intratracheal instillation of several concentrations of APC (0.2, 0.4, 1.0, 2.0 mg/kg of mouse body weight; n = 4) on the seventh day of BLM pump infusion. BALF sampling from each group of animals (n = 4) was performed on Day 21 after BLM infusion. Samples from the BLM/Veh and SAL/APC groups were also available for making comparison. Based on the results of these preliminary studies, APC was subsequently administered on the seventh day of BLM infusion and at a dose of 1 mg/kg of mouse body weight. BALF samples and lung tissue specimens were taken from each group of animals (n = 4) on Days 14 and 21 of BLM infusion.
Experimental Design
There were six treatment groups of animals (each with n = 5): (1) mice treated with sterile saline by minipump (SAL group), (2) mice treated with BLM by minipump (BLM group), (3) mice treated with saline by minipump and instillation of vehicle containing albumin (SAL/Veh group), (4) animals treated with BLM by minipump and instillation of vehicle with albumin 7 d after BLM administration (BLM/ Veh group), (5) mice treated with BLM by minipump and APC instillation 7 d after BLM administration (BLM/APC group), and (6) mice receiving saline by minipump and APC instillation 7 d after saline infusion.
Bronchoalveolar Lavage (BAL) and Blood Sampling
All animals were killed at defined time intervals by intraperitoneal injection of pentobarbital to take samples for biochemical and histologic examinations. Blood samples were collected by heart puncture
and placed in tubes containing one-tenth volume of 3.8% sodium citrate. BALF was obtained by cannulating the trachea with a 20-gauge
needle and infusing the lungs 4 times with 1 ml of saline. The recovery
of BALF ranged between 2.0 and 3.5 ml, with no significant differences in the volume recovered between control and treated mice. The
recovered fluid was filtered through a single layer of gauze to remove
mucus. The BALF was centrifuged (1,000 g, 10 min, 4° C) and the cell-free supernatant was stored immediately at 
80° C until use for biochemical analysis.
Histologic Examination and Fibrosing Score
After thoracotomy, the pulmonary circulation was flushed with saline and the lungs were removed. The left lung of each mouse was perfused with 10% neutral buffered formalin and fixed in formalin for 24 h. After embedding in paraffin, the tissue sections were prepared and stained with hematoxylin and eosin and examined by light microscopy. The Ashcroft scale was used for the quantitative histologic analysis of fibrotic changes induced by BLM (20). The severity of the fibrotic changes in each histologic section of the lung was assessed as a mean score of severity from observed microscopic fields. After examination of the whole section, the mean of the scores from all fields was taken as the fibrotic score. Each specimen was scored independently by four observers; finally, the mean of their individual scores was considered as the fibrotic score.
Hydroxyproline Assay
Collagen deposition was estimated by determining the total hydroxyproline content of the lung by high-performance liquid chromatography as described (21). Briefly, the right lung was excised, lyophilized, weighed, and then hydrolyzed in 6 N HCl at 110° C for 16 h in a sealed glass tube (Iwaki, Tokyo, Japan).
Biochemical Analysis of BALF
Protein concentration in BALF was measured by Bradford's method using a protein assay kit (Bio-Rad Laboratories, Hercules, CA) (22). The
activation of coagulation in plasma was evaluated by clotting time using
a KC-10 coagulometer (Heinrich Amelung, Amelung, Germany) as described previously (23). The BALF levels of thrombin and APC were
measured by amidolytic assay using the synthetic substrates S-2238 and
S-2366 (Chromogenix, Molndal, Sweden), respectively. The concentrations of thrombin and APC were extrapolated from curves drawn using
standard concentrations of thrombin and APC. The activity of plasminogen activator was spectrophotometrically measured using the chromogenic substrate S-2444 (Chromogenix, Molndal, Sweden). To measure the
balance between the coagulation system activation and fibrinolytic activity in the lung of the different treatment groups of animals, the ratio of
plasminogen activator activity to thrombin level in BALF was calculated. The BALF concentrations of TNF- and interleukin-1 (IL-1)
were measured using commercial mouse TNF- and IL-1 ELISA kits
(Biosource International, Camarillo,CA).
Reverse Transcriptase-Polymerase Chain Reaction
(RT-PCR) of TNF- and IL-1
Total RNA was extracted from lung tissue of the BLM/Veh and
BLM/APC groups of animals by the guanidine isothiocyanate procedure using Trizol Reagent (GIBCO, Grand Island, NY). A volume
of 5 µg of total RNA was reverse transcribed using oligo-dT primers and then the DNA was amplified by PCR. The RT-PCR was performed using the Superscript Preamplification System kit (Life Technologies, Gaithersburg, MD) following the manufacturer's instructions. The sequences of the primers used for TNF- complementary
DNA (cDNA) amplification were 5'-CCTGTAGCCCACGTCGTAGC-3' corresponding to 434 to 453 nucleotides and 5'-TTGACCTCAGCGCTGAGTTG-3' corresponding to 807 to 788 nucleotides and
for IL-1 5'-TATCAACCAACAAGTGATATTCTC-3' corresponding to 533 to 556 nucleotides and AGAAACAGTCCAGCCCATACTTT-3' corresponding to 908 to 886 nucleotides. PCR was performed
with 35 cycles, denaturation at 93° C for 30 s, annealing at 58° C for
30 s, and elongation at 73° C for 2 min. The cDNA of the housekeeping gene was amplified using primers contained in the kit provided by Superscript Preamplification System (Life Technologies, Gaithersburg, MD). The PCR products were separated on a 2% agarose gel containing 0.01% ethidium bromide, and the intensity of the stained bands was
quantitated by densitometric analysis on a Macintosh computer using
the public domain National Institutes of Health (NIH) image program
(Wayne Rasband, NIH, Research Service Branch, Bethesda, MD). The
amount of messenger RNA (mRNA) was normalized against the glyceraldehyde-3 phosphate dehydrogenase (GAPDH) mRNA.
Statistical Analysis
All data are expressed as mean ± SE, unless otherwise specified. The difference between the means of two variables was calculated by the Mann-Whitney U test and that between three or more variables by analysis of variance. A value of p < 0.05 was considered as statistically significant. Statistical analyses were carried out using the StatView 4.5 package for Macintosh (Abacus Concepts, Berkeley, CA).
| |
RESULTS |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
BALF Concentrations of Total Protein and Thrombin in Animals Treated with Intratracheal APC on Different Days after BLM Infusion
To determine the optimal timing for the treatment of BLM- induced lung injury with APC, the animals were treated with intratracheal instillation of APC on Days 3, 7, and 14 after BLM infusion and the concentrations of total protein and thrombin were measured in BALF samples taken on Day 21 of BLM infusion. As described in Figure 1, the concentrations of total protein and thrombin were significantly (p < 0.05) increased in mice of the BLM/Veh group compared with the SAL/Veh group. The concentrations of total protein and thrombin were significantly (p < 0.05) decreased in animals treated with APC (BLM/APC group) on Day 7 of BLM infusion compared with mice of the BLM/Veh group. No significant difference in total protein or thrombin was observed between the BLM/APC and BLM/Veh groups on Day 3 or 14. The concentrations of total protein and thrombin were not significantly different between the SAL/Veh and SAL/APC groups.
|
p < 0.05, compared with SAL/Veh group. *p < 0.05, compared with BLM/Veh group. n = 4 animals.
BALF Concentrations of Total Protein and Thrombin in Animals Treated with Different Doses of Intratracheal APC after BLM Infusion
To evaluate the dose-dependent effect of APC on BLM-induced lung injury, the animals were treated with several concentrations of APC on Day 7 after BLM infusion and the concentrations of total protein and thrombin were measured in BALF samples taken on Day 21 after BLM infusion. The concentrations of total protein and thrombin were significantly (p < 0.05) increased in mice treated with subcutaneous infusion of BLM and intratracheal instillation of Veh (BLM + Veh group) compared with animals treated with subcutaneous infusion of saline and intratracheal instillation of Veh (SAL + Veh group) (Figure 2). The concentrations of total protein were significantly (p < 0.05) decreased in animals treated with subcutaneous infusion of BLM and intratracheal instillation of APC (BLM + APC group) receiving 0.4, 1, or 2 mg of APC per kilogram of mouse body weight compared with mice of the BLM + Veh group. The concentration of thrombin was significantly (p < 0.05) decreased in animals of the BLM + APC group treated with 1 or 2 mg of APC per kilogram of mouse body weight compared with mice of the BLM + Veh group. No significant difference in BALF concentrations of total protein and thrombin was observed between animals treated with subcutaneous infusion of saline with intratracheal Veh (SAL + Veh group) and subcutaneous infusion of saline with intratracheal APC (SAL + APC group).
|
Total Protein and Markers of Clotting and Fibrinolysis Systems in BLM-Induced Lung Fibrosis
To measure the concentrations of total protein and markers of clotting and fibrinolysis systems, BALF samples and lung tissue specimens were collected on Days 3, 7, 14, and 21 after BLM infusion. As shown in Figure 3, the total protein concentration in BALF was significantly increased on Day 7 in mice treated with BLM alone and on Days 14 and 21 in mice treated with BLM and intratracheal instillation of vehicle (BLM/Veh) as compared with animals treated with pump infusion of saline alone (SAL group) or with pump infusion of saline with intratracheal instillation of vehicle (SAL/Veh), respectively. The thrombin concentration in BALF was significantly increased on Days 3 and 7 in the BLM group and on Days 14 and 21 in the BLM/Veh group of animals as compared with animals of the SAL and SAL/Veh groups, respectively (Figure 4). By contrast, APC concentrations in BALF from the BLM group were significantly reduced on Days 3 and 7 and in BALF from the BLM/Veh group on Days 14 and 21 as compared with animals of the SAL and SAL/Veh group, respectively (data not shown). The ratio of plasminogen activator activity to thrombin level in BALF from the BLM group on Day 7 and from the BLM/Veh group on Days 14 and 21 was significantly decreased as compared with animals of the SAL and SAL/Veh groups, respectively (Figure 5). Data from BLM groups on Days 14 and 21 are not shown. Overall, these findings show that BLM-induced lung injury is associated with increased intra-alveolar activation of the coagulation pathway with relatively decreased activity of the fibrinolysis system.
|
|
|
Effect of APC Intratracheal Instillation on Total Protein and Markers of Clotting and Fibrinolysis Systems in BLM-Induced Lung Fibrosis
To evaluate the effect of APC on BLM-induced lung injury, APC was intratracheally administered at a dose of 1 mg/kg of mouse body on Day 7 after BLM infusion (BLM/APC group). After intratracheal instillation of APC on Day 7, BALF samples and lung tissue specimens were collected on Days 14 and 21 after BLM pump infusion. The BALF concentrations of total protein (Figure 3) and thrombin (Figure 4) were significantly (p < 0.05) lower in the BLM/APC group than in animals of the BLM/Veh group. In addition, the ratio of plasminogen activator activity to thrombin level in BALF was significantly (p < 0.05) higher in the BLM/APC group than in animals of the BLM/ Veh group (Figure 5). There were no significant statistical differences in the BALF concentrations of total protein and thrombin and in the values of the plasminogen activator activity/thrombin ratio between SAL/Veh and SAL/APC groups of animals. APC concentrations in BALF remained unchanged on Day 14 but recovered moderately on Day 21 after APC treatment on Day 7 of BLM infusion (data not shown). Repeated doses (1 mg/kg body weight) of APC carried out on Days 3 and 7 after BLM administration did not further improve the BALF concentrations of total protein and thrombin compared with the improvement achieved with the single dose (1 mg/kg mouse body weight) of APC administered on Day 7 of BLM infusion (data not shown). The clotting time in plasma on Day 21 of BLM infusion was also measured to evaluate the effect of APC instillation on the systemic coagulation system; the clotting time in plasma was not different between the BLM/Veh and BLM/APC groups of animals (data not shown).
Histologic Findings and Markers of Collagen Deposition in BLM/Veh-treated and BLM/APC-Treated Mice
Lung injury in mice was induced by BLM infused using minipumps implanted subcutaneously and treated intratracheally with APC (BLM/APC group) or vehicle (BLM/Veh group) after 7 d of starting BLM administration. The intratracheal dosage of APC used in this study (1 mg/kg of mouse weight) was about approximately one-tenth of that intravenously used for treating lipopolysaccharide-induced lung vascular injury (23).
Fibrotic changes were progressive in mice treated with BLM/Veh after 14 (Figure 6A) and 21 (Figure 6B) days of treatment. Histologic examination after 14 d of BLM/Veh treatment showed focal fibrotic lesions mainly in subpleural and perivascular areas with consolidation of the lung parenchyma, loss of normal alveolar architecture, and increased infiltration of inflammatory cells in the interstitial and alveolar spaces. Twenty-one days after BLM/Veh treatment, lung fibrotic changes became more severe, expanding to the central regions of the lung parenchyma, involving the perivascular and peribronchiolar areas, and with more uniform areas of consolidation in subpleural regions of the lung. These histologic findings corresponded well with those observed in mice treated with BLM alone (without intratracheal administration of vehicle). Compared with the BLM/Veh group of animals, histologic findings on Days 14 (Figure 6C) and 21 (Figure 6D) in mice of the BLM/APC group showed less fibrotic lesions in the subpleural areas with central areas of lung parenchyma having only mild degree of cellular infiltration.
|
Quantitative Evaluation of Histologic Changes in BLM/Veh-treated and BLM/APC-treated Mice
The overall grade of lung fibrosis was assessed by the Aschcroft numerical score 21 d after BLM/Veh and BLM/APC treatment. There was a significant correlation between the fibrosis scores given by each observer (r = 0.9, p < 0.001). The mean fibrosis score in mice treated with BLM/Veh (4.5 ± 0.2) was higher than in mice treated with BLM/APC (2.3 ± 0.2). There was no significant difference in fibrosis score between mice treated with BLM and BLM/Veh. All mice survived until the termination of the experiment.
Collagen deposition in the lung was assessed by measuring the hydroxyproline content in the right lung of mice of each group. Compared with the SAL group, the hydroxyproline content of the lung significantly increased in the BLM/Veh group on Days 14 and 21 after BLM infusion (Figure 7). The hydroxyproline content was significantly higher (p < 0.05) in the lung of mice treated with BLM/Veh (1.78 ± 0.07 µmol/ lung weight) than in animals treated with BLM/APC (1.30 ± 0.06 µmol/lung weight) on Day 21 after BLM infusion (Figure 7). No significant differences in hydroxyproline content were observed between the SAL/Veh and SAL/APC groups.
|
Effect of Intratracheal APC Instillation on
Lung TNF- and IL-1 Expression
The effect of APC instillation on TNF- and IL-1 expressions in each group of animals was evaluated by enzyme immunoassays and RT-PCR. As shown in Figure 8, the concentrations of TNF- were significantly increased on Days 7 (BLM group) and 14 (BLM/Veh group) as compared with
control groups (SAL or SAL/Veh groups). The concentrations
of TNF- were significantly decreased in BALF of mice of the
BLM/APC group as compared with those observed in the
BLM/Veh group on Days 14 and 21 after BLM administration. The lung RNA expression of TNF-, as evaluated by
RT-PCR, was significantly decreased in the lungs of mice of
the BLM/APC group as compared with that observed in the
BLM/Veh group on the 14th and the 21st day after treatment
(data not shown). The concentrations of IL-1 were significantly higher (p < 0.05) in BALF from animals of the BLM/
Veh group than in BALF from the SAL/Veh group on Day 14 of BLM infusion (Figure 9). The concentrations of IL-1 were significantly decreased (p < 0.05) in BALF of mice of the
BLM/APC group as compared with the BLM/Veh group on
Day 14, but not on Day 21, after BLM administration. The
lung RNA expression of IL-1 in mice of the BLM/APC
group was significantly decreased only on Day 14 after BLM
administration (data not shown). These results indicate that
intratracheal administration of APC decreases the expression
and secretion of inflammatory cytokines in BLM-induced pulmonary fibrosis.
|
|
| |
DISCUSSION |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
It is well known that components of the coagulation and fibrinolysis pathways play important roles in processes of tissue injury and repair (24). Coagulation mechanisms are activated at sites of inflammation, and extravascular fibrin formation commonly occurs at foci of tissue injury (25). For example, alveolar fibrin deposition is prominent in most acute lung injuries and interstitial lung diseases (26). Lung damage induced by BLM is another good example of the importance of altered hemostasis in the development of tissue scar. After injury by BLM, lung epithelial and endothelial cells express tissue factor, which after binding to factor VIIa, activates the extrinsic coagulation pathway. Subsequently, the interstitium and alveolar spaces of the lung are filled up with plasma proteins and fibrin matrix (27). Under physiologic conditions, fibrin is rapidly degraded by the plasminogen-plasmin pathway, which is the main fibrinolytic system. However, when the fibrinolytic mechanism is impaired, increased intra-alveolar generation of thrombin occurs, leading to abnormal deposition of fibrin and collagen in the injured lung (27, 28). An important regulator of the fibrinolysis system is the PC pathway; APC, the enzyme effector of this pathway, inhibits coagulation activation and promotes fibrinolysis (10). In a previous study, we reported that clotting abnormalities during lung injury may be explained, at least in part, by dysfunction of the PC anticoagulant system (19). In the present study, we hypothesized that treatment with APC may block the development of fibrosis in BLM-induced lung injury.
Among available models of lung fibrosis, a continuous osmotic infusion pump was chosen because this model appears to mimic BLM-induced lung disease in patients more accurately than other model systems (2). Our present results showed for the first time that intra-alveolar thrombin formation is also remarkable in this model. Intratracheal administration was chosen for APC delivery because, in patients with interstitial lung disease, decreased PC activation in the intra-alveolar space is associated with plasma physiologic levels of PC activation markers (19). Intratracheal delivery has also the advantage of having less risk of causing hemorrhagic complications for excessive anticoagulation; this latter was also demonstrated in the present study for the lack of difference between the clotting times measured in plasma of BLM/Veh-treated and BLM/ APC-treated mice. APC was instilled on the seventh day of BLM treatment and immediately after withdrawing the BLM pump from the animals, because our preliminary experiments showed that lung inflammation and coagulation activation induced by BLM reach significant levels on the first week of BLM treatment; the BALF concentrations of total protein and thrombin were markedly increased on Day 7 after BLM infusion. Intratracheal instillation of APC on Day 7 was also found to improve lung injury, as indicated by the BALF concentrations of total protein and thrombin, more effectively than instillation on Days 3 or 14. Repeated doses of APC in early stages of lung injury (Days 3 and 7) were also carried out because the resolution process was previously demonstrated to depend on the severity and protraction of the early injury (29).
The significant decrease in the grade of lung fibrosis in mice treated with BLM/APC compared with mice treated with BLM/ Veh provides morphologic evidence of the inhibitory effect of APC on BLM-induced lung injury. The extensive pulmonary parenchyma consolidation in the central and subpleural regions together with intense fibrotic changes in peribronchiolar and perivascular areas observed in BLM/Veh-treated mice contrasted with the mild fibrotic changes detected in the BLM/ APC-treated mice. The marked decrease in the numerical fibrotic scoring and hydroxyproline content of the lung also supports the idea that APC inhibits lung fibrosis.
APC may inhibit lung fibrosis by different mechanisms. During the activation of the coagulation system, prothrombin is
converted to thrombin on cellular surface by the proteolytic
activity of the prothrombinase complex, an enzymic complex
composed of factor Xa, factor Va, phospholipids, and calcium
ions (30). Generated thrombin is known to stimulate the expression of proinflammatory cytokines and collagen from several cells and to induce migration and proliferation of fibroblast (31). APC inhibits the activation of coagulation system
by inactivating factor Va of the prothrombinase complex, and
thus blocks the generation of thrombin and the profibrotic effects of this protease (11). Another factor that favors fibrin deposition in the lung is the concurrent increase in local procoagulant activity and depression of fibrinolysis. A protracted
decrement in fibrinolytic activity of BALF has been described
in BLM-induced pulmonary fibrosis (24); this was corroborated in the present study by the decreased ratio of plasminogen activator activity to thrombin level observed in BALF of
the BLM-treated animals. This hypofibrinolysis is believed to
be mainly caused by increased intra-alveolar concentration of
PAI-1, which depresses local urokinase activity. APC may
neutralize PAI-1 activity and by this mechanism it may favor
the clearance of intra-alveolar fibrin by restoring the fibrinolytic activity to normal levels. Of great interest is the recent
report showing that APC per se may also exert anti-inflammatory effect (15, 16). APC has been described to inhibit the expression of proinflammatory cytokines from monocytes/macrophages, the migration of neutrophils, and the development
of vascular injury in lipopolysaccharide-induced sepsis. Upregulation of several proinflammatory cytokines has been associated with the occurrence of BLM-induced lung injury (32).
In the present study, the intratracheal instillation of APC was
also found to decrease the secretion of TNF- and IL-1 in
BALF from BLM-treated mice, suggesting that the inhibitory activity of APC on lung fibrosis may also be mediated, at least in part, by its anti-inflammatory effect.
In summary, the results of this study showed that intratracheal APC administration inhibits the development of lung fibrosis in BLM-induced lung injury and that, besides the anticoagulant effect of APC, its anti-inflammatory activity may also play an important role in the inhibition of lung fibrosis. These findings suggest a substantial therapeutic benefit of APC in BLM-induced lung injury. Further studies should be carried out to confirm the clinical efficacy of this therapy.
| |
Footnotes |
|---|
Correspondence and requests for reprints should be addressed to Dr. Esteban C. Gabazza, Mie University School of Medicine, Third Department of Internal Medicine, Edobashi 2-174, Mie 514-8507, Tsu City, Japan. E-mail: gabazza{at}clin.medic.mie-u.ac.jp
(Received in original form November 15, 1999 and in revised form October 4, 2000).
| |
References |
|---|
|
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Fleischmann RW,
Baber JR,
Thompson GR,
Schaeppi UH,
Illievsky VR,
Coonney DA,
Davis RD.
Bleomycin induced interstitial pneumonia in dogs.
Thorax
1971;
26:
675-682
2.
Harrison JH,
Lazo JS.
High dose continuous infusion of bleomycin in
mice: a new model for drug-induced pulmonary fibrosis.
J Pharmacol
Exp Ther
1987;
243:
1185-1194
3. Lindenschmidt RC, Tryka AF, Godfrey GA, Frome EL, Witschi H. Intratracheal versus intravenous administration of bleomycin in mice: acute effects. Toxicol Appl Pharmacol 1986; 85: 69-77 [Medline].
4. Theall RS, Barton RW. A comparision of lymphocyte population in lung tissue and in bronchoalveolar lavage fluid of rats at various times during the development of bleomycin-induced pulmonary fibrosis. Am Rev Respir Dis 1984; 170: 41-50 .
5. Olman MA, Mackman N, Gladson CL, Moser KM, Loskutoff DJ. Changes in procoagulant and fibrinolytic gene expression during bleomycin-induced lung injury in the mouse. J Clin Invest 1995; 96: 1621-1630 .
6.
Brown LF,
Asch B,
Harvey VS,
Buchinski B,
Dvorak HF.
Fibrinogen influx and accumulation of cross-linked fibrin in mouse carcinomas.
Cancer Res
1988;
48:
1920-1925
7. Gray AC, Bishop JE, Reeves JT, Mecham RP, Laurent GJ. Partially degraded fibrin(ogen) stimulates fibroblast proliferation in vitro. Am J Respir Cell Mol Biol 1995; 12: 684-690 [Abstract].
8.
Limper AH,
Roman J.
A versatile matrix protein with roles in thoracic
development, repair and infection.
Chest
1992;
101:
1663-1673
9. Kuhn C III,, Boldt J, King TE Jr,, Crouch E, Vartio T, McDonald JA. An immunohistochemical study of architectural remodeling and connective tissue synthesis in pulmonary fibrosis. Am Rev Respir Dis 1989; 140: 1693-1703 [Medline].
10. Suzuki K. Protein C in high KA. In: High KI, Robert HR, editors. Molecular basis of thrombosis and hemostasis. New York: Marcel Dekker; 1995.
11.
Esmon CT.
The regulation of natural anticoagulant pathways.
Science
1987;
235:
1348-1351
12. Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-1373 .
13. Comp PC, Esmon CT. Recurrent venous thromboembolism in patient with a partial deficiency of protein S. N Engl J Med 1984; 311: 1525-1528 [Abstract].
14.
Svensson PJ,
Dahlback B.
Resistence to activated protein C as a basis
for venous thrombosis.
N Engl J Med
1994;
330:
517-522
15. Grey ST, Tsuchida A, Hau H, Orthner CL, Salem HH. Selective inhibitory effects of the anticoagulant protein C on the responses of human mononuclear phagocytes to LPS, IFN-gamma, or phorbol ester. J Immunol 1994; 153: 3664-3672 [Abstract].
16.
Grinnell BW,
Herman RB,
Yan SB.
Human protein C inhibits selectin-mediated cell adhesion: role of unique fucosylated oligosaccharide.
Glycobiology
1994;
4:
221-225
17. Fukudome K, Esmon CT. Identification, cloning and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994; 42: 26486-26491 .
18. Hancock WW, Grey ST, Hau S, Akalin E, Orthner C, Sayegh MH, Salem HH. Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signalling and monocyte-dependent proliferative responses. Transplantation 1995; 60: 1525-1528 [Medline].
19.
Kobayashi H,
Gabazza EC,
Taguchi O,
Wada H,
Takeya H,
Nishioka J,
Yasui H,
Kobayashi T,
Hataji O,
Suzuki K,
Adachi Y.
Protein C anticoagulant system in patients with interstitial lung disease.
Am J Respir
Crit Care Med
1998;
157:
1850-1854
20.
Ashcroft T,
Simpson JM,
Timbrell V.
Simple method of estimating severity of pulmonary fibrosis on a numerical scale.
J Clin Path
1988;
41:
467-470
21. Woessner JF Jr.. The determination of hydroxyproline in tissue and protein samples containing small proportions of this amino acid. Arch Biochem Biophys 1961; 93: 440-447 [Medline].
22. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Ann Biochem 1976; 72: 248-254 .
23. Taylor FB Jr,, Chang A, Esmon CT, D'Angelo A, Vigano-D'Angelo S, Blick KE. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-925 .
24. Idell S. Coagulation, fibrinolysis, and fibrin deposition in lung injury and repair. In: SH Phan, RS Thrall, editors. Pulmonary fibrosis. New York: Dekker; 1995. p. 743-776.
25. Idell S, Peters J, James KK, Fair DS, Coalson J. Local abnormalities of coagulation and fibrinolytic pathways that promote alveolar fibrin deposition in the lungs of baboons with diffuse alveolar damage. J Clin Invest 1989; 84: 181-193 .
26. Chapman HA, Allen CL, Stone OL. Abnormalities in pathways of alveolar fibrin turnover among patients with interstitial lung disease. Am Rev Respir Dis 1986; 133: 437-443 [Medline].
27. Idell S, Gonzalez K, Gillies C, Fair DS, Thrall R. Abnormalities of pathways of fibrin turnover in lung lavage of rats with oleic acid and bleomycin-induced lung injury support alveolar fibrin deposition. Am J Pathol 1989; 135: 387-399 [Abstract].
28. Bertozzi P, Astedt BL, Zenzious L, Lynch F, LeMaire F, Zapol W, Chapman HA. Depressed bronchoalveolar urokinase activity in patients with adult respiratory distress syndrome. N Engl J Med 1990; 322: 890-897 [Abstract].
29. Burkhardt A. Alveolitis and collapse in the pathogenesis of pulmonary fibrosis. Am Rev Respir Dis 1989; 140: 513-524 [Medline].
30. Deguchi H, Takeya H, Gabazza EC, Nishioka J, Suzuki K. Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex. Biochem J 1997; 321: 729-735 .
31. Bar-Shavit R, Benezra M, Sabbah V, Bode W, Vlodavsky I. Thrombin as a multifunctional protein: induction of cell adhesion and proliferation. Am J Respir Cell Mol Biol 1992; 6: 123-130 .
32. Remick DG, DeForge LE. Cytokines and pulmonary fibrosis. In: SH Phan, RS Thrall, editors. Pulmonary fibrosis. New York: Dekker; 1995. p. 599-614.
This article has been cited by other articles:
 |
|
 |
|
  N Oikonomou, A Thanasopoulou, A Tzouvelekis, V Harokopos, T Paparountas, I Nikitopoulou, W Witke, A Karameris, A Kotanidou, D Bouros, et al. Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis Thorax, June 1, 2009; 64(6): 467 - 475. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. F. Mercer, R. H. Johns, C. J. Scotton, M. A. Krupiczojc, M. Konigshoff, D. C. J. Howell, R. J. McAnulty, A. Das, A. J. Thorley, T. D. Tetley, et al. Pulmonary Epithelium Is a Prominent Source of Proteinase-activated Receptor-1-inducible CCL2 in Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., March 1, 2009; 179(5): 414 - 425. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M R Looney, C T Esmon, and M A Matthay Role of coagulation pathways and treatment with activated protein C in hyperoxic lung injury Thorax, February 1, 2009; 64(2): 114 - 120. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. C. Chambers Abnormal wound healing responses in pulmonary fibrosis: focus on coagulation signalling Eur. Respir. Rev., December 1, 2008; 17(109): 130 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. M. du Bois and T. E. King Jr Elucidating the causes and examining the latest clinical findings in pulmonary fibrosis: recent progress and future objectives Eur. Respir. Rev., December 1, 2008; 17(109): 168 - 170. [Full Text] [PDF]
|
|
|
|
 |
|
 L. O. Mosnier, B. V. Zlokovic, and J. H. Griffin The cytoprotective protein C pathway Blood, April 15, 2007; 109(8): 3161 - 3172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chen, Z. Chen, N. R. Chintagari, M. Bhaskaran, N. Jin, T. Narasaraju, and L. Liu Alveolar type I cells protect rat lung epithelium from oxidative injury J. Physiol., May 1, 2006; 572(3): 625 - 638. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 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]
|
|
|
|
|
|
S. Weijer, C. W. Wieland, S. Florquin, and T. van der Poll A thrombomodulin mutation that impairs activated protein C generation results in uncontrolled lung inflammation during murine tuberculosis Blood, October 15, 2005; 106(8): 2761 - 2768. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. C.J. Howell, R. H. Johns, J. A. Lasky, B. Shan, C. J. Scotton, G. J. Laurent, and R. C. Chambers Absence of Proteinase-Activated Receptor-1 Signaling Affords Protection from Bleomycin-Induced Lung Inflammation and Fibrosis Am. J. Pathol., May 1, 2005; 166(5): 1353 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Yuda, Y. Adachi, O. Taguchi, E. C. Gabazza, O. Hataji, H. Fujimoto, S. Tamaki, K. Nishikubo, K. Fukudome, C. N. D'Alessandro-Gabazza, et al. Activated protein C inhibits bronchial hyperresponsiveness and Th2 cytokine expression in mice Blood, March 15, 2004; 103(6): 2196 - 2204. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. W. Rijneveld, S. Weijer, S. Florquin, C. T. Esmon, J. C. M. Meijers, P. Speelman, P. H. Reitsma, H. Ten Cate, and T. van der Poll Thrombomodulin mutant mice with a strongly reduced capacity to generate activated protein C have an unaltered pulmonary immune response to respiratory pathogens and lipopolysaccharide Blood, March 1, 2004; 103(5): 1702 - 1709. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Idell The Matrix Unloaded: Aerosolized Heparin or Urokinase for Pulmonary Fibrosis Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1268 - 1269. [Full Text] [PDF]
|
|
|
|
|
|
A. Gunther, N. Lubke, M. Ermert, R. T. Schermuly, N. Weissmann, A. Breithecker, P. Markart, C. Ruppert, K. Quanz, L. Ermert, et al. Prevention of Bleomycin-induced Lung Fibrosis by Aerosolization of Heparin or Urokinase in Rabbits Am. J. Respir. Crit. Care Med., December 1, 2003; 168(11): 1358 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.C. Chambers Role of coagulation cascade proteases in lung repair and fibrosis Eur. Respir. J., September 20, 2003; 22(44_suppl): 33s - 35s. [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
S. Shimizu, E. C. Gabazza, O. Taguchi, H. Yasui, Y. Taguchi, T. Hayashi, M. Ido, T. Shimizu, T. Nakagaki, H. Kobayashi, et al. Activated Protein C Inhibits the Expression of Platelet-derived Growth Factor in the Lung Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1416 - 1426. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Tobin Compliance (COMmunicate PLease wIth Less Abbreviations, Noun Clusters, and Exclusiveness) Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): 1534 - 1536. [Full Text] [PDF]
|
|
|
|
|
|
E. C. Gabazza, O. Taguchi, Y. Adachi, E. C. Gabazza, O. Taguchi, and Y. Adachi BLEOMYCIN-INDUCED LUNG FIBROSIS: THE AUTHORS SHOULD HAVE USED ANOTHER METHOD TO INDUCE PULMONARY LESIONS RESEMBLING HUMAN IDIOPATHIC PULMONARY FIBROSIS Am. J. Respir. Crit. Care Med., March 15, 2002; 165(6): 845b - 846. [Full Text]
|
|
|
|
|
|
M. J. TOBIN Tuberculosis, Lung Infections, Interstitial Lung Disease, and Socioeconomic Issues in AJRCCM 2001 Am. J. Respir. Crit. Care Med., March 1, 2002; 165(5): 631 - 641. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Proc. Am. Thorac. Soc. | Am. J. Respir. Cell Mol. Biol. |