Urokinase

Bleomycin is a well known fibrogenic agent, provoking an initial adult respiratory distress syndrome-like injury with subsequent strong fibroproliferative response. Severe abnormalities of the alveolar surfactant system, which may be linked to the appearance of alveolarfibrin deposition, have been implicated in the pathogenetic sequence of events. Using a model of standardized aerosol delivery of 1.8 U bleomycin/kg body weight in rabbits, we investigated the influence of repetitive nebulization of heparin or urokinase-type plasminogen activator (u-PA) on the development of lung fibrosis. In an "early" (Days 2-12 postbleomycin) or "late" (Days 14-24 postbleomycin) treatment protocol, approximately 3,500 U heparin or approximately 6,500 U u-PA was delivered to the bronchoalveolar space. Within four weeks, the bleomycin challenge provoked severe pulmonary fibrosis with reduction of lung compliance, marked increase in soluble collagen (bronchoalveolar lavage fluid) and hydroxyproline content (lung tissue), a typical reticular fibrosis pattern on high-resolution computed tomography, and typical histologie findings. Therapeutic intervention resulted in a far-reaching normalization of compliance, suppression of soluble collagen and hydroxyproline accumulation, and virtual abrogation of the computed tomography scan and histologie features of lung fibrosis, with most prominent effects seen in the early heparin and late u-PA administration. No bleeding complications occurred. These findings strongly support the concept that alveolar fibrin generation is an important event in the development of postbleomycin lung fibrosis. "Compartmentalized" anticoagulation and/or fibrinolysis via inhalational deposition of interventional agents in the alveolar compartment may thus offer a new therapeutic strategy for prevention of fibrosis.

Keywords: fibrinolysis; pulmonary surfactant; coagulation; interstitial lung disease; diffuse parenchymal lung disease

Idiopathic pulmonary fibrosis represents a frequent and, unfortunately, still unresolved clinical issue (1,2). The underlying pathogenetic events are largely unsettled, and treatment options are limited, mostly including corticosteroids and azathioprine (3). Recently, some new therapeutic strategies have been developed, aiming to modulate the cytokine and growth factor balance (4), to increase antioxidant capacities (5), and to prevent collagen deposition, e.g., by use of proline analogs (6). Encouraging clinical data were obtained from a pilot study investigating the effects of the antifibrogenic cytokine intcrferon-[gamma] (7).

Further insights into the pathophysiology underlying fibroproliferative lung disorders stem from the recent observation that, next to the changes in the cytokine and growth factor balance, abnormalities of the pulmonary surfactant system may be involved in the sequence of events. In idiopathic pulmonary fibrosis, the surface activity of pulmonary surfactant is severely impaired, and profound alterations of its biochemical composition are encountered (8-10). In addition, inhibition of surfactant function by plasma-derived proteins has been shown to occur in vitro (11), under conditions of severe inflammatory injury with vascular leakage (12,13) and also in interstitial lung disease (8). Fibrinogen leakage may be particularly relevant under these conditions: its surfactant inhibitory capacity is further increased by approximately two orders of magnitude on conversion to fibrin, and a nearly complete "incorporation" of all hydrophobic surfactant compounds into the arising fibrin matrix has been documented as the underlying mechanism (14). In vitro, surface activity can be restored by induction of fibrinolysis, yielding liberation of surface-active material from the fibrin lattice and, thus, decrease of surface tension (15). u-PA and tissue-type plasminogen activator were particularly effective as therapeutic agents for such "rescue" of surface activity (16). Interestingly, it has been shown that the alveolar hemostatic balance, although being antithrombotic and profibrinolytic under regular conditions, is shifted toward the prothrombotic side with increased procoagulant and decreased fibrinolytic capacities in the bronchoalveolar compartment under conditions of acute (e.g., adult respiratory distress syndrome [ARDS] [17, 18]) and chronic inflammatory lung diseases (e.g., idiopathic pulmonary fibrosis [19-21]). These abnormalities include markedly increased tissue factor and factor VII (FVII) activities and elevated plasminogen activator inhibitor-1 and [alpha]^sub 2^-antiplasmin levels in the alveolar lining layer, and the dramatically augmented D-dimer concentration in the bronchoalveolar lavage fluid (BALF) of patients with ARDS and idiopathic pulmonary fibrosis is a direct indicator of the overall increased alveolar fibrin turnover in these prototype diseases of acute and chronic lung inflammation.

How may surfactant abnormalities and coagulation disorders in the alveolar compartment contribute to lung fibrosis? Loss of surface tension-lowering properties at the alveolar fluid-air interface is known to result in alveolar instability (22). Alveolar fibrin formation as the most potent of surfactant inhibitory mechanisms may cause atelectasis. In addition, histologic studies suggested fibrin-mediated apposition ("gluing") of alveolar septae as a general feature in lung fibrosis of different etiology (23). According to this concept of "collapse induration," the fibrin matrix is a nidus for fibroblast invasion, resulting in scarring and thereby irreversible loss of alveoli, with traction of the remaining airspaces (honeycombing). Moreover, direct fibroblast-activating properties have been shown for thrombin (24,25) and fibrin(ogen) scission products (26).

This study was undertaken to investigate the role of alveolar fibrin formation in the development of lung fibrosis in an animal model. Aerosolization of a standardized dose of bleomycin was used to provoke a sequence of initial acute lung injury with subsequent strong fibroproliferative response in rabbits. In this model, protein leakage, increased alveolar procoagulant activities, hyaline membrane formation, and severe deterioration of surfactant function are part of the pathogenetic sequence. Four weeks after bleomycin nebulizalion, severe pulmonary fibrosis became evident from loss of lung compliance, typical computed tomography (CT)-scan abnormalities and histologie findings, and a marked increase in soluble collagen (lavage) and lung hydroxyproline content. In the current study, aerosolization of either heparin or u-PA, undertaken during the development of fibrosis to directly target the alveolar coagulation processes without exerting changes in the systemic hemostatic balance, resulted in a far-reaching reduction of fibrosis, as documented by physiological, biochemical, CT-scan, and histologie parameters. No side effects such as bleeding were noted in these studies. These findings thus (1) strongly support the concept that alveolar fibrin generation is an important event in the development of lung fibrosis in response to bleomycin and (2) offer targeted intervention in the alveolar hemostatic balance as a therapeutic strategy for prevention of lung fibrosis.

METHODS

For a detailed description of METHODS see online supplement.

Lung Fibrosis Model

Healthy White New Zealand rabbits received 1.8 U/kg body weight bleomycin via ultrasonic nebulization under mechanical ventilation at Day 0. All animal protocols were approved by the Justus Liebig University's Committee on Animal Investigations.

Experimental Croups

Saline solution, unfractionated heparin, or human recombinant u-PA were each administered repetitively by ultrasonic nebulization (mass median devodynamic diameter 2.73 µm via a tightly fitting mask and under spontaneous breathing. Pilot studies in healthy rabbits ascertained (1) persistent elevation (~ 48 hours) of recalcification times in BALF on aerosol delivery of the heparin dosage as used in this study and (2) induction of fibrinolysis at the alveolar level in the absence of any increase in epithelial and cndothclial permeabilities (see also online supplement). Furthermore, in additional pilot experiments using bleomycinchallenged rabbits, aerosolization of the currently used dose of u-PA provoked a switch in the alveolar hemostatic balance, with increased fibrinolytic capacity. Similarly, aerosolization of the currently used dose of heparin resulted in a reversal of the procoagulant response (for details see online supplement). The following groups were investigated (1) control (n = 11): no bleomycin, sham-aerosolization with saline; (2) bleomycin (n = 9): inhalation of bleomycin, no intervention; (3) bleomycin plus early heparin treatment (n = 7); pulmonary deposition of 3,510 ± 117 U heparin/nebulization maneuver at Days 2, 4, 6, 8, 10, and 12 postbleomycin challenge; (4) bleomycin plus late heparin treatment (n = 7): pulmonary deposition of 3,572 ± 117 U heparin/ nebulization maneuver at Days 14, 16, 18, 20, 22, and 24 postbleomycin challenge; (5) bleomycin plus early u-PA treatment (n = 5): pulmonary deposition of 6,319 ± 26.5 U u-PA/nebuli/ation maneuver at Days 2, 4, 6, 8,10, and 12 postbleomycin challenge; and (6) bleomycin plus late u-PA treatment (n = 7): pulmonary deposition of 6,889 ± 12.3 U u-PA/ nebulization maneuver at Days 14, 16, 18, 20, 22, and 24 postbleomycin challenge.

Examination of Lungs at Day 28

On intubation and mechanical ventilation of the animals at Day 28, a high-resolution CT was performed. Afterward, arterial blood specimen was obtained, and static lung compliance was calculated from the linear part of the deflation limb and with respect to the esophageal pressure and the animal weight. After application of an overdose of KelanestRompun, lungs were excised, ventilated ex vivo, and perfused with an artificial buffer system as described recently (27). The capillary filtration coefficient (given in cm^sup 3^sec/mm Hg/g wet lung weight × 10^sup -4^) and the total vascular compliance were determined gravimetrically. Finally, ventilation was stopped at the end of inspiration, the left main bronchus was ligated, and the right lung was lavaged (BAL) using warm saline (3 × 30 ml). Afterward, the left pulmonary artery was cannulated with a small catheter, and formaline was instilled al a constant pressure of 25 cm H2O. Next, the left lung was separated and embedded in Paraplast plus (Sigma, Munich, Germany), cut into 4-µm slices, and stained with hcmatoxylin-eosin according to standard techniques. The right lung was then carefully liberated from major bronchi and vessels, homogenized, and stored for analysis of hydroxyproline content (28). BAL cells were sedimented (300 × g, 4°C, 10 minutes), stained, and counted according to standard techniques. Cell-free BAL was analyzed for soluble collagen by reaction with Sirius Red and for surface activity on isolation of crude surfactant pellets (48,000 × g, 1 hour, 4°C) by means of a pulsating bubble surfactometer (13). The surface tension after 12 seconds of film adsorption ([gamma]^sub ads^) is given.

Statistical Analysis

Values are represented as mean ± SE. Statistical differences (significance level 0.05) between the various groups were evaluated using the H test first, followed by pairwise comparison using the Mann-Whitney U test. Significance level is indicated by *or ^sup +^ (p

RESULTS

Nebulization of 1.8 U bleomycin/kg body weight in intact rabbits provokes an acute lung injury with maximum gas exchange abnormalities being noted after 4 days (data not given in detail). Gas exchange then largely recovers during the subsequent fibroproliferative period, but is not fully resolved, as evidenced from the decreased Pa^sub o2^ values measured 28 days after bleomycin aerosolization in the present study (Figure 1). At this time point, however, a marked lung stiffening was noted, as depicted in Figure 2, the static lung compliance, assessed in the intact animals, was found to be 3.5 ± 0.29 ml/mm Hg/kg body weight in the sham-aerosolized controls but only 1.8 ± 0.23 ml/mm Hg/kg body weight in the bleomycin-challenged animals. In accordance, the peak inspiratory pressure, measured during standardized mechanical ventilation after earlier isolation of the lungs from the thorax, was highly significantly increased in the postbleomycin lungs (Figure 3). Moreover, an increase in endothelial permeability was detected in these lungs, with capillary filtration coefficient values being approximately threefold increased as compared with the saline-aerosolized controls (Figure 4) and with an increased alveolar protein load being measured in the BALF. In addition, surface activity of large surfactant aggregates was significantly impaired (Table 1). In contrast, there was only marginally increased leukocyte invasion into the alveolar compartment at this late time point after bleomycin nebulization (Table 1). Peripheral blood cell counts were fully unchanged, documenting absence of any significant myelosuppressive effect of this mode of inhalational bleomycin administration (Table 2).

CT scans (Figure 5) and histologie examination (Figure 6) revealed extensive fibrosis in virtually all lung regions in the bleomycin-exposed rabbits, with some predominance of the basal lung areas. In contrast, fully normal pictures were obtained in the animals undergoing sham aerosolization with saline 28 days before examination. The abnormalities in the postbleomycin high-resolution CT were mostly characterized by a fine reticular pattern, with no major ground glass opacities. In analogy, hematoxylin-eosin stains of the lungs (Figure 6) showed markedly increased extracellular matrix surrounding fibroblasts and some capillaries mostly at the alveolar level, sometimes also with subpleural and interlobular localization. Next to these developing scars, enlarged alveolar sacculi with loss of septae and bronchiolization were observed. There were no inflammatory exudates or major infiltration with leukocytes at this late time point after bleomycin nebulization.

In accordance with the loss of lung compliance and the fibrotic changes demonstrated in the high-resolution CT and the histology findings, the soluble collagen concentrations in the BALF and the hydroxyproline content of lung tissue were markedly elevated in the postbleomycin lungs when compared with the saline controls (Figures 7 and 8).

All interventions addressing coagulation processes in the alveolar compartment (early and late-aerosolized heparin, early and late-aerosolized u-PA) were well tolerated by the animals, and there was no evidence for bleeding complications from the daily examination of the animals, from histologic investigation of the lung tissue, or from measurements of hemoglobin and hematocrit. Most impressively, therapeutic interventions significantly reduced the development of physiologic dysfunction, as evidenced from (1) improved lung compliance (Figure 2), (2) decreased peak inspiratory pressure on standardized ex vivo ventilation (Figure 3), (3) reduction of the reticular pattern in the CT scans (Figure 5), (4) decrease of the histologie abnormalities including extracellular matrix deposition and fibroblast appearance (Figure 6), and (5) marked reduction of soluble collagen in the BALF and hydroxyproline in lung tissue (Figures 7 and 8). Moreover, the capillary filtration coefficient and alveolar protein levels were nearly normalized in all lungs from heparin- or urokinase-treated animals (Figure 4), and surface activity was improved (Table 1). The attenuation of the fibroproliferative response to the bleomycin challenge was most obvious on early heparin nebulizalion and late u-PA aerosolization (Figures 2, 3, and 5-7). No significant influence of any of these interventions was encountered in view of the cell differential (Table 1) and recalcification times (data not given in detail).

DISCUSSION

In the present study, aerosol technology was used to ensure homogenous distribution of bleomycin to rabbit lungs via the inhalational route. Previous detailed investigations in this model showed that the nebulized dose of 1.8 U/kg body weight of this fibrogenic agent provokes an initial ARDS-like type of injury, with gas exchange abnormalities being most prominent after 4 to 8 days, characterized by manifold enhanced endothelial permeability, a marked influx of leukocytes and protein leakage into the alveolar space, enhanced procoagulant activity in the BALF, and a pronounced deterioration of the alveolar surfactant system (data not given in detail). Alveolar edema, hyaline membrane formation, and atelectasis are prominent histologic findings during this early response to bleomycin. This is followed by a strong fibroproliferativc response, being the key abnormality of the currently investigated 4-week period after bleomycin challenge. Pulmonary iibrosis, homogenously distributed within the lung, with some accentuation of the basal regions, which may be due to preferred aerosol distribution to these areas, was documented by a severe loss of lung compliance, increased soluble collagen in the BALF and lung hydroxyproline content, a typical reticular fibrosis pattern in the CT scan, and the histologic finding of fibroblast invasion and matrix deposition. Some increase in endothclial permeability was still noted, reflected by the elevated capillary filtration coefficient values, and there was a moderate impairment of arterial oxygenation at this postbleomycin time point. The homogeneity of the fibrotic response evoked by the aerosol delivery of bleomycin compares favorably with the mode of intratracheal bleomycin instillation, where predominance of peribronchial and peribronchiolar fibrosis is noted in particular in larger animals (29).

The pathogenetic mechanisms underlying the rapid (ibroproliferative response to bleomycin are still not completely understood. Relevant features include a complex scenario of cellular recruitment (29, 30), activation of cytokines (31, 32) and growth factors (33-36), and cross talk between these proinflammatory and proproliferative pathways. Moreover, disturbances of the alveolar surfactant system have been noted in postbleomycin lungs, similar to those in human interstitial lung disease (8, 37-41). When analyzed for the present model of bleomycin acrosolization, these abnormalities were shown to include a marked loss of surface tension-lowering properties on biophysical characterization, decreased percentages of the surface-active large surfactant aggregate fraction, reduction in key surfactant components such as dipalmitoylphosphatidylcholine and the apoproteins B and C, as well as presence of surfactant-inhibitory proteinaceous material in the alveolar compartment (data not given in detail).

Against this background, the present investigation focused on the role played by alveolar fibrin generation in the pathogenesis of postbleomycin lung fibrosis. As in human idiopathic pulmonary fibrosis, increased alveolar fibrin formation due to fibrinogen leakage, elevated procoagulant activities (mostly tissue factor and FVII mediated), and depressed antifibrinolytic capacities have been demonstrated in blcomycin-induced lung injury (42-44). The approach chosen to address this issue in the present study was the compartmentalized administration of heparin for prevention of coagulation processes and of u-PA for scission of librin(ogen) products. Repetitive aerosol delivery in spontaneously breathing animals was undertaken for both agents, with protocols allowing either "early" or "late" intervention with these tools. The dose of heparin inhaled per nebulization maneuver was approximately 3,500 U: pilot experiments with titration of nebulized heparin showed that this dosage caused a significant inhibition of coagulation in subsequently assessed BALF in both healthy as well as bleomycin-challenged lungs, whereas no systemic anticoagulative effect of heparin was noted (see Table 1 in the online supplement). Interestingly, the anticoagulalive effect of aerosolized heparin in the alveolar compartment persisted for at least 36 hours, as previously suggested (45). Concerning the u-PA doses (~ 6,500 U being deposited in the bronchoalveolar space within one aerosolization maneuver), pilot studies in isolated perfused rabbit lungs had shown that this dosage of the protease did not elicit any change in endo- and epithelial barrier properties, whereas local fibrinolytic activity was documented on reassessment of BALF (46). In intact animals, no systemic fibrinolytic effect was observed after alveolar deposition of this relatively low amount of u-PA. In bleomycin-challenged rabbits, this dosage was shown to reverse the antifibrinolytic capacity in the alveolar compartment into a more profibrinolytic state (see Figure 1 in the online supplement). In line with these pilot studies, no evidence for systemic bleeding complications was obtained on use of heparin and u-PA in the present investigation. Notably, this was even true for the lung itself, as enhanced blood entry into the alveolar spaces was not observed on histologic examination of the lungs. Thus, the repetitive aerosol delivery of the doses of heparin and u-PA used in this study was found to be suitable to restrict the effect to the alveolar compartment and to be safe with respect to bleeding complications in the model of bleomycin-induced lung fibrosis.

Most impressively, all variables indicating the development of lung fibrosis were markedly affected by both heparin and u-PA treatment. This was first true for parameters that might be directly linked with the absence or presence of alveolar fibrin, i.e., the lung compliance (assessed in the intact animals) and the peak inspiratory pressure (measured during standardized mechanical ventilation of the isolated lungs). Via surfactant inhibition, as discussed previously, and loss of recruitable alveolar spaces due to fibrin gluing, alveolar fibrin accumulation might directly contribute to the marked reduction of compliance in the bleomycin-treated animals. It is, nevertheless, a notable observation that the optimum treatment regimens (early heparin and late u-PA) fully normalized both the compliance and the peak inspiratory pressure in the postbleomycin lungs. Second, the variables reflecting fibroblast activation and related matrix deposition were significantly altered by both inhalational heparin and u-PA. The lavage levels of soluble collagen were significantly suppressed, the lung tissue hydroxyproline content was virtually normalized, the key histologic changes of increased appearance of fibroblasts with surrounding extracellular matrix were clearly reduced, and the CT-scan picture of a fine relicular fibrosis pattern was largely prevented. Third, the optimum treatment regimens (early heparin and late u-PA) were also found to attenuate the otherwise still increased epithelial and endothelial permeability and to restore surface activity within the alveolar compartment. This is of interest as (1) these agents thus apparently did not induce lung vascular leakage when being deposited in the alveolar compartment, a possibility to be considered when using aerosolized u-PA, and (2) such a beneficial impact on the lung barrier properties may suppress fibrosis by limiting intraalveolar access of further protein (and fibrinogen), which is consistent with the lowered protein levels in the BALF. It is currently unknown how interference with alveolar coagulation processes may influence the lung barrier properties. However, improved surface tension characteristics at the alveolar surface may be an important link, as severe surfactant abnormalities may per se promote lung edema formation ("high-surface tension pulmonary edema") (47, 48).

Our observations are thus fully in line with the concept that alveolar fibrin formation is an important trigger event "linking" acute inflammatory injury and fibroproliferative responses. It may not be clearly deduced from this study whether this effect of "compartmentalized" anticoagulation proceeds via blockage of thrombin and fibrin(ogen) scission products as soluble fibroblast stimulating factors (24-26) or whether the suppression/removal of fibrin matrices serving as nidus for fibroblast invasion (23) is the more relevant mechanism. Heparin aerosolization will interfere with both sequelae, and it is well in accordance with the time course of the increased procoagulant activity in bleomycin-challenged lungs (maximum after 4-16 days, see Figure 3 in the online supplement) (42-44) that early heparin was more efficient than the late administration of this anticoagulant agent. However, the strong efficacy of late u-PA, which is expected to split fibrin clots but does not interfere with thrombin activity and will even liberate fibrin(ogen) scission products, favors the assumption that the removal of fibrin matrices from the alveolar spaces per se represents a most important aspect of the beneficial impact of the presently undertaken anticoagulative/fibrinolytic interventions. Considering the efficacy of u-PA, it is of interest that in vitro studies showed rescue of intact surfactant from fibrin polymers incorporating the hydrophobic surfactant compounds on incubation with u-PA (16). The restoration of surface activity in the treated animals observed in this study further supports the assumption that the currently undertaken therapeutic steps indeed resulted in improved alveolar surface tension regulation and thus in alveolar reopening. In view of the hydroxyprolin and soluble collagen data, it may be further speculated whether collagen deposition as such may be better addressed by an earlier u-PA treatment protocol. Further studies are warranted to more closely elucidate this question.

Moreover, the present findings are well in line with recent studies in transgenic mice lacking or overexpressing plasminogen activator inhibitor-1, in which the extent of fibrosis in response to bleomycin was shown to be linked to the amount of available plasminogen activator inhibitor-1 (49). In this model system, relative overexpression of plasminogen activator inhibitor-1 potentiated the fibrotic response, whereas absence of plasminogen activator inhibitor-1 attenuated the response. In another study, Howell and colleagues (50) reported that direct thrombin inhibition reduced lung collagen accumulation in a rat bleomycin model of pulmonary fibrosis and suggested that thrombin may act via both effects, its classical role in the coagulation cascade (i.e., fibrin formation) and its cellular effects mediated by its major cellular receptor, protease-activated receptor-1 (51). In line with these findings, protein C activity, which indirectly controls thrombin generation by inactivation of the clotting factors FVa and FVIIIa (tenase and prothrombinase complex), was found to be reduced and associated with abnormal collagen turnover in patients with interstitial lung disease (52). Consequently, the same group showed that intratracheal application of activated protein C in a mouse model of bleomycin-induced lung fibrosis was capable of inhibiting the development of fibrosis (53). Furthermore, systemic heparin administration was previously also found to cause some attenuation of lung hydroxyproline content in bleomycin-induced fibrosis in mice (54). In a mouse model of intratracheal bleomycin administration, Sisson and coworkers (55) observed a reduction of Day 28 hydroxyproline values but not of histologic appearance on intratracheal application of an adenoviral vector encoding human u-PA at Day 21. In parallel experiments, the same vector encoding murine u-PA turned out to be ineffective. The reasons for their inconsistent results are presently not clear. Major aspects do, however, include the relatively late application time point (with an earlier administration of the vector being impossible due to superimposed inflammatory events and thus increased mortality of the mice) and the differing degree of u-PA activity being generated by the two vectors. In view of the present data, it is nevertheless clear, that u-PA application may prevent or even reverse experimental lung fibrosis when administered timely and in suitable amounts. Finally, the very recent observation that bleomycin application may result in increased lung hydroxyproline content also in fibrinogen-deficient mice, does not necessarily contradict this interpretation of our data (56).

In conclusion, both aerosolized heparin (maximum efficacy when administered in the early postbleomycin period) and nebulized u-PA (strongest efficacy when applied in the later course) nearly completely suppressed the fibroproliferative response of rabbit lungs to bleomycin challenge. This included normalization of lung compliance, suppression of soluble collagen and hydroxyproline accumulation, and virtual abrogation of the CT scan and histologic features of lung fibrosis. The interventions using aerosol technology were designed to exert anticoagulative/profibrinolytic effects in the alveolar compartment without alterations of the systemic hemostatic balance. The findings strongly support the view that alveolar fibrin generation is an important event in the development of lung fibrosis in response to bleomycin treatment and that compartmentalized anticoagulation or fibrinolysis via inhalational deposition of agents in the alveolar compartment may offer a new therapeutic regimen for prevention of lung fibrosis triggered by acute type lung injury.

Conflict of Interest Statement: A.G. has no declared conflict of interest; N.L. has no declared conflict of interest; M.E. has no declared conflict of interest; R.T.S. has no declared conflict of interest; N.W. has no declared conflict of interest; A.B. has no declared conflict of interest; P.M. has no declared conflict of interest; C.R. has no declared conflict of interest; K.Q. has no declared conflict of interest; L.E. has no declared conflict of interest; F.G. has no declared conflict of interest; W.S. has no declared conflict of interest.

References

1. Coultas DB, Zumwalt RE, Black WC, Sobonya RE. The epidemiology of interstitial lung diseases. Am J Respir Crit Care Med 1994;150:967-972.

2. Phan SH. New strategies for treatment of pulmonary fibrosis. Thorax 1995;50:415-421.

3. Mason RJ, Schwarz MI, Hunninghake GW, Musson RA. Pharmacological therapy for idiopathic pulmonary fibrosis: past, present, and future. Am J Respir Crit Care Med 1999;160:1771-1777.

4. Nakao A, Fujii M, Matsumura R, Kumano K, Saito Y, Miyazono K, Iwamoto T. Transient gene transfer and expression of Smad7 prevents blcomycin induced lung fibrosis in mice. J Clin invest 1999;104:5-11.

5. Parizada B, Werber MM, Nimrod A. Protective effect of human recombinant MnSOD in adjuvant arthritis and bleomycin-induced lung fibrosis. Free Radic Res Commun 1991;15:297-301.

6. Greco MJ, Kemnitzer JE, Fox JD, Choe JK, Kohn J, Riley DJ, Poiani GJ. Polymer of proline analogue with sustained antifibrotic activity in lung fibrosis. Am J Respir Crit Care Med 1997;155:1391-1397.

7. Zische R, Hofbauer E, Wittnann K, Petkov V, Block LH. A preliminary study of long-term treatment with interferon gamma 1-b and low-dose prednisolon in patients with idiopathic pulmonary fibrosis. N Engl J Med 1999;341:1264-1269.

8. Gunther A, Schmidt R, Nix F, Yabut-Perez M, Guth C, Rosseau S, Siebert C, Grimminger F, Morr H, Velcovsky HG. Surfactant abnormalities in idiopathic pulmonary fibrosis, hypersensitivity pneumonitis and sarcoidosis. Eur Respir J 1999;14:565-573.

9. Robinson PC, Watters LC, King TE, Mason RJ. Idiopathic pulmonary fibrosis: abnormalities in bronchoalveolar lavage fluid phospholipids. Am Rev Respir Dis 1988;137:585-591.

10. McCormack FX, King TE, Voelker DR, Robinson PC, Mason RJ. Idiopathic pulmonary fibrosis: abnormalities in bronchoalveolar lavage content of surfactant protein A. Am Rev Respir Dis 1991;144:160-166.

11. Fuchimukai T, Fujiwara T, Takahashi A, Enhorning G. Artificial pulmonary surfactant inhibited by proteins. J Appl Physiol 1987;62:429-437.

12. Kobayashi T, Kitta K, Ganzuka M, Inui S, Grossmann G, Robertson B. Inactivation of exogenous surfactant by pulmonary edema fluid. Pediatr Res 1991;29:353-356.

13. Gunther A, Siebert C, Schmidt R, Ziegler S, Grimminger F, Yabut M, Temmesfeld B, Walmrath D, Morr H, Seeger W. Surfactant alterations in severe pneumonia, ARDS and cardiogenic lung edema. Am J Respir Crit Care Med 1996;153:176-184.

14. Seeger W, Elssner A, Gunther A, Kramer HJ, Kalinowski HO. Lung surfactant phospholipids associate with polymerizing fibrin: loss of surface activity. Am J Respir Cell Mol BM 1999;9:213-220.

15. Gunther A, Kalinowski M, Elssner A, Seeger W. Clot embedded natural surfactant: kinetics of fibrinolysis and surface activity. Am J Physiol 1994;267:L618-L624.

16. Gunther A, Markart P, Kalinowski M, Ruppert C, Grimminger F, Seeger W. Cleavage of surfactant-incorporating fibrin by different fibrinolytic agents: kinetics of lysis and rescue of surface activity. Am J Respir Cell Mol BM 1999;21:738-745.

17. Idell S, Koenig KB, Fair DS, Martin TR, McLarty J, Maunder RJ. Serial abnormalities of fibrin turnover in evolving adult respiratory distress syndrome. Am J Physiol 1991;261:L240-L248.

18. Gunther A, Mosavi P, Heinemann S, Ruppert C, Muth H, Markart P, Grimminger F, Walmrath D, Temmesfeld-Wollbruck B, Seeger W. Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia: comparison with the respiratory distress syndrome. Am J Respir Crit Care Med 2000;161:454-462.

19. de Moerloose P, de Benedetti E, Nicod L, Vifian C, Reber G. Procoagulant activity in bronchoalvcolar lavage fluids: no relationship with tissue factor pathway inhibitor activity. Thromb Res 1992;65:507-518.

20. Gunther A, Mosavi P, Ruppert C, Heinemann S, Temmesfeld-Wollbruck B, Velcovsky HG, Morr H, Grimminger F, Walmrath D, Seeger W. Enhanced tissue factor pathway activity and fibrin turnover in the alveolar compartment of patients with interstitial lung disease. Thromb Haemost 2000;83:853-860.

21. Kotani I, Sato A, Hayakaya H, Urano T, Takada Y, Takada A. Increased procoagulant and antifibrinolytic activities in the lungs with idiopathic pulmonary librosis. Thromb Res 1995;77:493-504.

22. Griese M. Pulmonary surfactant in health and human lung diseases: state of the art. Eur Respir J 1999;13:1455-1476.

23. Burkhardt A. Alveolitis and collapse in the pathogenesis of pulmonary fibrosis. Am Rev Respir Dis 1989;140:513-524.

24. Ohba T, McDonald JK, Silver RM, Strange C, Carwile LeRoy E, Ludwicka A. Scleroderma bronchoalveolar lavage fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of platelet-derived growth factor [alpha]-receptor. Am J Respir Cell Mol Biol 1994;10:405.

25. Hernandez-Rodriguez NA, Harrison NK, Chambers RC, Gray AJ, Aotuhcott AM, du Bois RM, Black CM, Scully MF, McAnulty RJ. Role of thrombin in pulmonary fibrosis. Lancet 1995;346:1071-1073.

26. Gray AJ, Bishop JE, Reeves JT, Mecham RP, Laurent GJ. Partially degraded fibrin(ogen) stimulates fibroblast proliferation in vitro. Am J Respir Mol Cell Biol 1995;12:684-690.

27. Seeger W, Walmrath D, Grimminger F, Rosseau S, Schutte H, Kramer HJ, Ermert L, Kiss L. Adult respiratory distress syndrome: model systems using isolated perfused rabbit lungs. In: Packer L, editor. Methods in enzymology. Vol. 233: oxygen radicals in biological systems. San Diego: Academic Press; 1994. p. 549-584.

28. Berg RA. Determination of 3- and 4-hydroxyproline. In: Cunnighman LW, Frederiksen DW, editors. Methods in enzymology. Vol. 82: structural and contractile proteins. San Diego: Academic Press; 1982. p. 372-398.

29. Thrall RS, McCormick JR, Jack RM, McReynolds RA, Ward PA. Bleomycin-induced pulmonary librosis in the rat: inhibition by indomethacin. Am J Pathot 1979;95:117-130.

30. Jones HA, Schofield JB, Krausz T, Boobis AR, Haslelt C. Pulmonary fibrosis correlates with duration of tissue neutrophil activation. Am J Respir Crit Care Med 1998;158:620-628.

31. Piguet PF, Haufman S, Barazzone C, Muller M, Ryffel B, Eugster HP. Resistance of TNF/LT alpha double deficient mice to bleomycininduced fibrosis. Int J Exp Pathol 1997;78:43-48.

32. Zhang K, Gharaee-Kermani M, McGarry B, Remick D, Phan SH. TNF-alpha mediated lung cytokine networking and eosinophil recruitment in pulmonary fibrosis. J Immunol 1997;158:954-959.

33. Lasky JA, Ortiz LA, Tonthat B, Hoyle GW, Corti M, Athas G, Lungarella G, Brody A, Friedman M. Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis. Am J Physiol 1998;275:L365-L371.

34. Coker RK, Laurent GJ, Shahzeidi S, Lympany PA, du Bois RM, Jefferey PK, McAnulty RJ. Transforming growth factors-beta 1, -beta 2, and -beta 3 stimulate fibroblast procollagen production in vitro but are differentially expressed during bleomycin induced lung fibrosis. Am J Pathol 1997;150:981-991.

35. Temelkovski J, Kumar RK, Maronese SE. Enhanced production of an EGF-like growth factor by parenchymal macrophages following bleomycin-induced pulmonary injury. Exp Lung Res 1997;23:377-391.

36. Gurujeyalakshmi G, Hollinger MA, Giri SN. Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level. Am J Physiol 1999;276:L311-L318.

37. Robinson PC, Watters LC, King TE, Mason RJ. Idiopathic pulmonary fibrosis: abnormalities in bronchoalveolar lavage fluid phospholipids. Am Rev Respir Dis 1988;137:585-591.

38. McCormack FX, King TE, Voelker DR, Robinson PC, Mason RJ. Idiopathic pulmonary fibrosis: abnormalities in bronchoalveolar lavage content of surfactant protein A. Am Rev Respir Dis 1991;144:160-166.

39. Thrall RS, Swendsen CL, Shannon TH, Kennedy CA, Frederick DS, Grunze MF, Sulavik SB. Correlations of changes in pulmonary surfactant phospholipids with compliance in bleomycin-induced pulmonary fibrosis in the rat. Am Rev Respir Dis 1987;136:113-118.

40. Osanai K, Takahashi K, Sato S, Iwabuchi K, Ohtake K, Sata M, Yasui S. Changes of lung surfactant and pressure-volume curve in bleomycininduced pulmonary fibrosis. J Appl Physiol 1991;70:1300-1308.

41. Horiuchi T, Mason RJ, Kuroki Y, Cherniack RM. Surface and tissue forces, surfactant protein A, and the phospholipid components of pulmonary surfactant in bleomycin-induced pulmonary fibrosis in the rat. Am Rev Respir Din 1990;141:1006-1013.

42. Idell S, Gonzales KK, MacArthur CK, Gillies C, Walsh PN, McLarty J, Thrall RS. Bronchoalveolar lavage procoagulant activity in bleomycininduced lung injury in marmorsets: characterization and relationship to fibrin deposition and fibrosis. Am Rev Respir Dis 1987;136:124-133.

43. 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.

44. Olman MA, Simmons WL, Pollman DJ, Loftis AY, Bini A, Miller EJ, Fuller GM, Riviera KE. Polymerization of fibrinogen in murine bleomycin-induced lung injury. Am J Physiol 1996;271:L519-L526.

45. Kohler D. Aerosols for systemic treatment. Lung 1990;168:677-684.

46. Schermuly RT, Gunther A, Ermert M, Ermert L, Ghofrani HA, Weissmann N, Grimminger F, Seeger W, Walmrath D. Conebulization of surfactant and urokinase restores gas exchange in perfused lungs with alveolar fibrin formation. Am J Physiol Lung Cell Mol Physiol 2001;280:L792-L800.

47. Nieman GF, Goyette D, Paskanik A, Brendenberg CH. Surfactant displacement by plasma lavage results in pulmonary edema. Surgery 1990;107:677-683.

48. Nieman GF, Brendenberg CE. High surface tension pulmonary edema induced by detergent aerosol. J Appl Physiol 1985;58:129-136.

49. Eitzman DT, McCoy RD, Zheng X, Fay WP, Shen T, Ginsburg D, Simon RFI. Bleomycin-induced pulmonary fibrosis in transgenic mice that either lack or overexpress the murine plasminogen activator inhibitor-1 gene. J Clin Invest 1996;97:232-237.

50. Howell DC, Goldsack NR, Marshall RP, McAnulty RJ, Starke R, Purely G, Laurent GJ, Chambers RC. Direct thrombin inhibition reduces lung collagen, accumulation, and connective tissue growth factor mRNA levels in bleomycin-induced pulmonary fibrosis. Am J Pathol 2001;159:1383-1395.

51. Howell DC, Laurent GJ, Chambers RC. Role of thrombin and its major cellular receptor, protease-activated receptor-1, in pulmonary fibrosis. Biochem Soc Trans 2002;30:211-216.

52. Yasui H, Gabazza EC, Taguchi O, Risteli J, Risteli L, Wada H, Yucla H, Kobayashi H, Suzuki K, Adachi Y. Decreased protein C activation is associated with abnormal collagen turnover in the intraalveolar space of patients with interstitial lung disease. Clin Appl Thromb Hemost 2000;6:202-205.

53. Yasui H, Cabazza EC, Tamaki S, Kobayashi T, Hataji O, Yuda H, Shimizu S, Suzuki K, Adachi Y, Taguchi O. Intratracheal administration of activated protein C inhibits bleomycin-induced lung fibrosis in the mouse. Am J Respir Cru Care Med 2001;163:1660-1668.

54. Piguet PF. Van GY, Guo J. Heparin attenuates blcomycin but not silicainduced pulmonary fibrosis in mice: possible relationship with involvement of myofibroblasts in bleomycin, and fibroblasts in silica-induced fibrosis. Int J Exp Pathol 1996;77:155-161.

55. Sisson TH, Hattori N, Xu Y, Simon RH. Treatment of bleomcyin-induced pulmonary fibrosis by transfer of urokinase-type plasminogen activator genes. Hum Gene Ther 1999;10:2315-2323.

56. Hattori N, Degen JL, Sisson TH, Liu H, Moore BB, Pandrangi RG, Simon RH, Drew AF. Bleomycin-induced pulmonary fibrosis in fibrinogen-null mice. J Clin invest 2000;106:1341-1350.

Andreas Gunther, Norbert Lubke, Monika Ermert, Ralph T. Schermuly, Norbert Weissmann, Andreas Breithecker, Philipp Markart, Clemens Ruppert, Karin Quanz, Leander Ermert, Friedrich Grimminger, and Werner Seeger Departments of Internal Medicine, Anatomy, Radiology, and Pathology, Justus-Liebig University, Giessen, Germany

(Received in original form January 29, 2002; accepted in final form August 14, 2003)

Supported by Deutsche Forschungsgemeinschaft (DFC), GU 405/3-1.

Correspondence and requests for reprints should be addressed to Andreas Gunther, M.D., Department of Internal Medicine, Med. Klinik II Klinikstrasse 36, D-35385 Giessen, Germany. E-mail: andreas.guenther@innere.med.uni-giessen.de

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Am J Respir Crit Care Med Vol 168. pp 1358-1365, 2003

DOI: 10.1164/rccm.2201082

Internet address: www.atsjournals.org

Copyright American Thoracic Society Dec 1, 2003
Provided by ProQuest Information and Learning Company. All rights Reserved