Departments of 1 Internal Medicine, 2 Anatomy, and 3 Pathology, Justus-Liebig-University Giessen, D-35392 Giessen, Germany
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ABSTRACT |
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Alveolar fibrin
generation has been suggested to possess strong surfactant-inhibitory
potency. In perfused rabbit lungs, fibrin formation in the alveolar
space was induced by sequential ultrasonic aerosolization of fibrinogen
and thrombin, and the efficacy of rescue administration of surfactant
and urokinase was investigated. Ventilation-perfusion
(A/
) distribution was assessed by the multiple inert gas elimination technique. Aerosolization of fibrinogen (~20 mg/kg body wt) increased shunt flow to ~7%. Sequential
nebulization of fibrinogen and thrombin (1.3 U/kg body wt) caused
alveolar fibrin deposition, documented immunohistologically, and
provoked marked shunt flow, progressing to ~22% at the end of the
experiments. The hemodynamics were virtually unchanged. Rescue
aerosolization of natural bovine surfactant (15 mg/kg body wt) or
urokinase-type plasminogen activator (4,500 U/kg body wt), undertaken
after fibrin formation, improved gas exchange but progressive shunt
flow still occurred (efficacy, surfactant > urokinase). In
contrast, conebulization of surfactant and urokinase reversed shunt
flow to ~7%, with an increased appearance of normal
A/
matching. We conclude that alveolar fibrin
formation is a potent surfactant-inhibitory mechanism in intact lungs,
provoking severe
A/
mismatch with a
predominance of shunt flow, and that rescue aerosolization of
surfactant plus urokinase may offer restoration of gas exchange under
these conditions.
urokinase-type plasminogen activator; thrombin; fibrinogen; shunt flow; aerosolization; ventilation-perfusion ratio
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INTRODUCTION |
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ALVEOLAR FIBRIN FORMATION is a hallmark of many inflammatory lung diseases including acute respiratory distress syndrome (ARDS) (13, 16). Under these conditions, both a manyfold increased leakage of plasma proteins, including fibrinogen, across a leaky endothelial and epithelial barrier and a pronounced shift of the alveolar hemostatic balance toward the procoagulative side have been documented (1, 10, 15, 16). Alveolar protein levels may be elevated >10-fold in ARDS lungs, and the procoagulant activity, attributable to tissue factor and factor VII, was noted to be increased by approximately two orders of magnitude in severe ARDS and pneumonia. Both alveolar macrophages and epithelial cells have been shown to express and shed tissue factor into the alveolar lining layer in response to inflammatory stimuli (3, 14). Moreover, alveolar levels of the urokinase-type plasminogen activator (uPA), the predominant plasminogen activator of the bronchoalveolar compartment, were found to be depressed in ARDS lungs (1, 10, 16), whereas plasminogen activator inhibitor-1 levels were highly increased under these conditions (4, 15, 21). Both fibrinogen leakage and these alterations in the alveolar hemostatic balance may thus be assumed to underlie alveolar fibrin formation in ARDS and other acute inflammatory lung diseases.
Pulmonary surfactant is a lipoprotein complex covering the alveolar
surface and reducing the alveolar surface tension to near zero values
at end expiration. Thereby, atelectasis during end expiration is
prevented, and breathing is made feasible under regular transthoracic
pressures. Impaired surface activity, with minimum surface tension
values of 20 mN/m, has consistently been observed in surfactant
isolates from patients with ARDS (6, 10, 11) or
severe pneumonia, necessitating mechanical ventilation (10). These changes have been ascribed to a variety of
biochemical alterations, including changes in the phospholipid, fatty
acid, and surfactant apoprotein profiles. In addition, inhibition of surfactant function by plasma proteins leaked into the alveolar space
has been established as a powerful surfactant-inhibitory mechanism
(10). An in vitro study (28) documented the
surfactant-inhibitory capacity to be fibrinogen > hemoglobin > albumin. Moreover, the generation of a fibrin clot from
fibrinogen in the presence of pulmonary surfactant was shown to cause
the most severe surfactant inhibition in another in vitro study
(27). Compared with (soluble) fibrinogen, the
dose-inhibition curve of fibrin is shifted to the left by more than two
orders of magnitude. The incorporation of hydrophobic surfactant
compounds into the nascent fibrin matrix, with a concomitant severe
loss of surface tension-lowering properties, has been suggested as the
underlying mechanism. Interestingly, in vitro proteolysis of
surfactant-incorporating fibrin clots was shown to be suitable for
"rescue" of the surfactant material trapped in the fibrin matrix
(8), with its surface tension-lowering properties being conserved.
The present study employed aerosol techniques for sequential alveolar
delivery of fibrinogen and thrombin in intact rabbit lungs to create a
model of selective alveolar fibrin formation. Marked deterioration of
ventilation-perfusion (A/
) matching with a
predominance of shunt flow, reminiscent of gas exchange disturbances in
ARDS, occurred under these conditions, with changes far surpassing
those of sole fibrinogen challenge. Rescue inhalation of either
urokinase or surfactant, undertaken after formation of the alveolar
fibrin clots, reduced the gas exchange abnormalities, but by far the
most effective approach for restoration of gas exchange properties was
the combined rescue administration of surfactant and urokinase.
Targeting alveolar fibrin formation by transbronchial administration of
surfactant plus urokinase might thus offer a new therapeutic strategy
in acute inflammatory lung diseases with extensive hyaline membrane
formation, thereby further expanding current strategies of
transbronchial surfactant administration in patients with severe ARDS
(7, 32).
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METHODS |
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Materials.
The mouse monoclonal anti-human D-dimer antibody (murine IgG3 clone
DD-3B6/22) was purchased from American Diagnostic (Greenwich, CT). The
antibody recognizes D-dimer and cross-linked fibrin derivatives (DD-E)
and shows no reactivity with human fibrinogen or fibrinogen degradation
products. Bovine serum albumin (BSA-C) and silver-enhancer solution
were purchased from Biotrend (Cologne, Germany). Human uPA (Actosolv;
specific activity 120,000 U/mg protein), thrombin (specific activity
1,250 U/mg protein), and fibrinogen (Haemocomplettan) were from Behring
(Marburg, Germany). Bovine surfactant was a gift from Dr. Karl Thomae
(Alveofact, Boehringer Ingelheim, Biberach an der Riss, Germany).
Isolated lung model. The perfused lung model has been previously described in detail (29). Briefly, rabbits were deeply anesthetized and anticoagulated with heparin (1,000 U/kg). After a tracheostomy, the animals were ventilated with room air. After a midsternal thoracotomy, catheters were placed into the pulmonary artery and left atrium, and perfusion with Krebs-Henseleit buffer was started. The lungs were perfused (total volume 400 ml) with a flow of 150 ml/min. Left atrial pressure was set at 1.0 mmHg in all experiments. In parallel with the onset of artificial perfusion, room air supplemented with 5% CO2 was used for ventilation (volume-controlled, positive end-expiratory pressure 1 cmH2O). The isolated lungs were then ventilated and perfused for a steady-state period lasting 30 min. Lungs that displayed visible signs of infection, gained >2 g of weight during the steady state, or showed an increase in pulmonary arterial pressure (Ppa) were excluded from the further course of the study.
Gravimetric estimation of the capillary filtration coefficient. The capillary filtration coefficient (Kfc) was determined gravimetrically, employing a sudden venous pressure elevation of 10 cmH2O for 8 min. Kfc was calculated by time (t) 0 extrapolation of the slope of the weight gain with a semilogarithmic plot of the rate of weight gain of the lung according to Taylor and Gaar (30). Vascular compliance was determined as the change in vascular volume per change in microvascular pressure. The initial rapid change in weight over the first 2 min was used for the calculation of vascular compliance (30). Retention weight was defined as the difference in weight gain before and after venous pressure elevation.
Aerosol procedures. Aerosolization of isotonic saline, human uPA, human fibrinogen (15 mg/ml), and a calf lung surfactant extract (15 mg/ml) was performed by means of an ultrasonic nebulizer (Pulmo Sonic 5500, DeVilbiss Medizinische Produkte, Langen, Germany). This device produces an aerosol with a mass median aerodynamic diameter of 4.5 µm and a geometric standard deviation of 2.1 as measured with a laser diffractometer (HELOS, Sympatec, Clausthal-Zellerfeld, Germany). Bovine thrombin was aerosolized with another ultrasonic nebulizer (Portasonic II, DeVilbiss Medizinische Produkte), with a mass median aerodynamic diameter of 4.3 µm and a geometric standard deviation of 2.2. The nebulizers were connected to the inspiratory tubing of the ventilation system. Condensation of aerosolized material within the inspiratory limb of the ventilation system was reduced by heating to 40°C. Under these conditions, a lung deposition rate of 25% of the initially generated aerosol mass was achieved (25). This deposition fraction, which was controlled periodically, was taken into consideration when calculating the quantity of agents delivered to the bronchoalveolar space by aerosolization. The total nebulized dose was obtained by weighing the nebulizer before and after the aerosolization maneuvers. In a preceding study (25), ultrasonic nebulization was ascertained not to impair the biophysical and biochemical properties of surfactant material. Similarly, the clotting ability of the fibrinogen preparation used in the present study was found to be unaffected by the aerosolization procedure (>95% clot formation both pre- and postnebulization). In contrast, the specific activities of uPA and thrombin were found to be reduced to 89 and 73%, respectively, of the initially provided activity (measured in vitro by aerosol trapping and subsequent measurement of activity by means of the chromogenic substrates S-2444 and S-2238). All data concerning the total deposited pulmonary activity of thrombin and urokinase were corrected for this loss of activity during the nebulization procedure.
A/
determination in isolated lungs by
multiple inert gas elimination technique.
The continuous
A/
distributions were
determined by the multiple inert gas elimination technique (MIGET) as
described by Wagner and West (31). Determination
of
A/
distribution was performed by least
squares analysis. An indication of the acceptable quality of the
A/
distributions is a residual sum of squares of 5.348 or less in half of the experimental runs (50th percentile) or
10.645 or less in 90% of the experimental runs (90th percentile) (31). In the present study, 72.4% of the residual sum of
squares were <5.348 and 98.4% were <10.645.
Experimental protocol.
Seven experimental groups were employed. The treatment protocol of six
groups is shown in Fig. 1. At the end of
the steady-state period, a baseline A/
measurement was performed and time was set at zero.
Nebulization of the different agents was then undertaken from
t = 0 until t = 60 min, from
t = 65 until t = 75 min, and from
t = 95 until t = 155 min. Additional
MIGET analysis was performed at 90, 165, and 195 min. The control group
received nebulized saline to ensure equivalent fluid load of the lungs.
In the fibrinogen (Fbg) group, aerosolization of fibrinogen was
combined with two saline nebulization periods, resulting in a total
fluid loading of 7.4 g as measured for this group. In the fibrin
(Fbg-Thr) group, fibrinogen was aerosolized with subsequent
nebulization of thrombin to induce fibrin formation. Total lung fluid
deposition was 8.6 g. Rescue applications were undertaken after
the preceding fibrin formation, with the timing and dose of these
rescue applications shown in Fig. 1. The protocols included the
administration of surfactant (Fbg-Thr-Surf group), uPA (Fbg-Thr-uPA
group), and a combination of these agents (Fbg-Thr-Surf-uPA group). The
total lung fluid load measured in these experiments was 7.0, 7.4, and 7.3 g, respectively.
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Bronchoalveolar lavage.
Bronchoalveolar lavage (BAL) was performed at the end of each
experiment. For this purpose, a total volume of 50 ml of saline was
instilled and reaspirated three times, and the recovered fluid was
filtered through sterile gauze. Cells were removed by centrifugation at
300 g for 10 min, and the cell-free supernatant was divided into aliquots and stored at 80°C until further processing.
Quantification of D-dimer. D-dimer was measured by a commercially available ELISA kit (Boehringer Mannheim, Mannheim, Germany). The assay was performed as recommended by the supplier.
Immunohistochemistry and microscopy. Fixation was performed by overnight immersion of the lungs in a 3% paraformaldehyde solution (n = 3 lungs). For paraffin embedding, whole lungs were dissected in tissue blocks from all lobes. Sectioning at 10 µm was performed from all paraffin-embedded blocks. Immunohistochemistry was performed as recently described in detail (5). Briefly, paraffin sections were dewaxed, rehydrated, and preincubated in PBS containing 5% goat serum, 0.025% BSA-C, 0.05% Tween 20, and 0.02 M glycine to block unspecific binding. Overnight incubation with the monoclonal anti-human D-dimer antibody diluted 1:50 was carried out at 4°C. Incubation with secondary gold-conjugated antibodies diluted 1:400 was performed overnight at 4°C. Next, the sections were fixed for 5 min in 2% phosphate-buffered glutaraldehyde and after several washes in glass double-distilled water, incubated in silver enhancer solution for 50 min. Counterstaining of the sections was performed with nuclear fast red. Control staining was performed by omission of the primary antibody or substitution with nonspecific preimmune serum at the same dilution.
Microscopy was performed with a Leica DM RXA microscope (Leica, Wetzlar, Germany) at a magnification of ×250. Epipolarization depiction of the immunogold-silver-stained structures created a complete segmentation between positively stained and nonstained tissue. Gray-scale images were digitized with a 12-bit cooled charge-coupled device camera (Sensys KAF 1400, Photometrics, Tucson, AZ) connected with the host computer. For direct visualization of staining intensity, a pseudocolor scale with 11 colors was chosen, each representing an equal sector of the intensity scale, and applied to the images.Data analysis. All values are given as means ± SE or as a coefficient of variation (SD/mean in percent). For analysis of statistical differences, two-tailed Student's t-test for unpaired samples was performed. After Bonferroni's correction, a P value < 0.05 was considered significant.
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RESULTS |
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Baseline variable responses to the nebulization of saline.
All lungs employed in the current study displayed Ppa values in the
range between 6 and 9 mmHg and a mean Kfc value
of 5.2 × 105 ± 1.2 × 10
5
ml · s
1 · cmH2O
1 · g
lung wet weight
1 at the end of the steady-state period.
Baseline
A/
measurements revealed a unimodal,
narrow distribution of perfusion and ventilation to midrange
A/
(0.1 <
A/
< 10) areas throughout. Shunt flow
(
A/
< 0.005) and perfusate flow to
poorly ventilated areas (low
A/
areas;
0.005 <
A/
< 0.1) were extremely low (<2.5%), and no perfusion of high
A/
regions (10 <
A/
< 100) was
observed (data not shown in detail). Dead space ventilation (
A/
> 100) approximated 50% of
ventilation in this system of isolated lung ventilation. These data
correspond to previous investigations in the perfused rabbit lung
(29) and document a physiological
A/
distribution and an intact endothelial and/or epithelial barrier in these isolated organs.
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Vascular permeability and gas exchange in response to inhaled
uPA and thrombin.
Nebulization of 9.4 U/kg body wt of thrombin did not affect Ppa, weight
gain, ventilation pressure (Table 2), or
lung gas exchange (data not shown in detail).
Kfc, vascular compliance, and retention (weight
gain during venous pressure challenge) remained unchanged (Table 2).
Similarly, lung deposition of 4,111 ± 564 U uPA/kg body wt did
not alter Kfc, vascular compliance, retention, and Ppa (Table 2). However, when uPA doses of 6,106 ± 889 and 10,227 ± 2,031 U/kg body wt were provided to the alveolar
compartment, significant increases in Kfc values
up to 11.9 × 105 ± 3.6 × 10
5 and 22.2 × 10
5 ± 2.9 × 10
5
ml · s
1 · cmH2O
1 · g
lung wet weight
1, respectively, were noted (Table 2). The
highest uPA dose also caused a marked increase in fluid retention
(weight gain) during the venous challenge maneuver. Ppa, vascular
compliance, and ventilation pressure remained unchanged.
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Effects of fibrinogen deposition and fibrin formation in the alveolar compartment. Aerosolization of fibrinogen resulted in a moderate increase in shunt flow from 1.2 ± 0.3 to 7.4 ± 2.1% and a slight increase in perfusion to poorly ventilated areas to 1.3 ± 1.1% (Table 1). Dead space ventilation increased by ~5%. Ppa remained constant (7.2 ± 0.4 mmHg at 0 min and 7.4 ± 0.4 mmHg at 195 min), whereas lung weight displayed a progressive increase, resulting in a total weight gain of 10.7 ± 1.8 g at the end of the experiments. As in the control group, the major part of this weight gain was, however, due to the total deposited aerosol mass of 7.4 g.
Induction of fibrin formation by sequential aerosol application of fibrinogen and thrombin caused severe deterioration of
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Rescue administration of surfactant and/or uPA.
In lungs with preceding fibrin formation due to the combined
application of fibrinogen and thrombin, aerosol application of surfactant resulted in a significant decrease in shunt flow to 12.1 ± 2.7%. The percentage of perfusion of low
A/
areas was largely unchanged by this
intervention (5.4 ± 3.2%; Table 1). Perfusion of normal
A/
areas was thus increased compared with that in the Fbg-Thr group (Fig. 3). Dead space ventilation
was 63.4% at the end of the experiments.
A/
mismatch, indicated by log SD
, was reduced compared with that in the Fbg-Thr group (Table 1). Ppa was not altered by this
intervention. Total lung weight gain was 18.2 ± 1.8 g at the
end of the experiments (7 g of deposited fluid volume) in the
Fbg-Thr-Surf group, and some increase in ventilation pressure occurred.
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BAL fluid analysis.
Measurement of the BAL fluid D-dimer levels showed very low
concentrations of this scission product of cross-linked fibrin in the
control and fibrinogen-treated lungs (Fig.
5). Subsequent to alveolar fibrin
formation due to additional thrombin aerosolization (Fbg-Thr group),
the BAL fluid D-dimer content increased significantly, and this also
held true for the surfactant-treated (Fbg-Thr-Surf) group. The highest
D-dimer levels in the BAL fluid were encountered in the two uPA
treatment (Fbg-Thr-uPA and Fbg-Thr-Surf-uPA) groups.
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DISCUSSION |
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The present study was performed in a model of
buffer-perfused, ventilated rabbit lungs previously
characterized in detail (29). The advantages of this model
are 1) control of pulmonary hemodynamics, continuous
monitoring of lung weight, and repetitive assessment of the
Kfc, 2) the absence of bloodborne
components that might possibly confound the effects caused by selective
alveolar fibrin formation, and 3) the suitability to employ
aerosol technology for alveolar drug delivery. In addition, MIGET,
previously adapted to this isolated lung model (33),
offers a powerful tool to analyze gas exchange conditions in detail
(23). As documented by baseline measurements,
hemodynamics, endothelial and/or epithelial barrier function, and
A/
matching are well within the physiological range in these buffer-perfused lungs.
In the control experiments, nebulization of saline was undertaken to
mimic the extent of alveolar fluid deposition effected by the
experimental protocols employing fibrinogen aerosolization. Interestingly, the net lung fluid loading of 8 g, assessed both by
measurement of aerosolized mass and by monitoring of lung weight gain,
did not affect pulmonary hemodynamics, vascular integrity, and
ventilation pressure at all. Moreover, physiological
A/
matching was virtually fully maintained,
with only minimal shunt flow (
3%) noted at the end of the
experiments. Thus alveolar fluid deposition per se, resulting in an
approximate doubling of the baseline lung weight, is apparently well
tolerated with respect to gas exchange in these intact lungs.
Alveolar deposition of a fibrinogen solution, again causing an ~8-g
net lung weight, provoked an increase in shunt flow to 7% along
with a very minor increase in perfusion of low
A/
areas and a corresponding decrease in
perfusion of normal
A/
regions. This finding
is very compatible with an in vitro study (28) that
demonstrated the surfactant-inhibitory capacities of fibrinogen. When
the ratio of aerosolized fibrinogen (~20 mg/kg body wt) to the
endogenous rabbit alveolar surfactant pool [~7 mg/kg
(25)] is calculated, some moderate inhibition of
surfactant function is to be anticipated from the in vitro studies, and
this might well cause some limited extent of atelectasis and shunt flow.
In vitro studies addressing the surfactant-inhibitory capacity of
proteins also forwarded the concept that hydrophobic surfactant components, phospholipids, and the surfactant apoproteins B and C are
incorporated into a nascent fibrin matrix and that nearly complete
incorporation of these surfactant constituents, with severe inhibition
of surface activity, already occurs at a fibrin-to-surfactant ratio of
1:5 (27). This study is the first one to rigorously test
this concept in intact lungs by sequential aerosolization of fibrinogen
and thrombin, the latter being applied at a dose that per se does not
induce pulmonary abnormalities. With this approach, the generation of
up to ~20 mg/kg body wt of fibrin was to be anticipated, and the
appearance of alveolar fibrin deposits, very much resembling hyaline
membrane formation, was nicely demonstrated by the immunohistological
studies. Indeed, a marked progressive shunt flow, surpassing 20% at
the end of the experiments, as well as perfusion of low
A/
areas (4-5%) was noted in these
lungs, indicating severe surfactant deterioration, with a loss of
alveolar patency in the fibrin-loaded lungs. Concomitantly, the
perfusion of normal
A/
areas was
significantly decreased. Thus fibrin generation in the alveolar
compartment is apparently a potent surfactant-inhibitory mechanism,
with a severe impact on gas exchange under conditions of intact lung
architecture. This finding also supports previous investigations
(22, 24, 25) employing different surfactant-inhibitory
interventions in intact lungs (e.g., lavage, detergents, acid
aspiration) in which a deterioration of
A/
matching with a predominance of
shunt flow was consistently demonstrated.
In several of these previously reported models of surfactant
inhibition (18-20, 24, 25), transbronchial
administration of exogenous surfactant either by instillation or by
aerosolization was found to improve gas exchange and, in particular, to
reduce shunt flow, compatible with restoration of alveolar surfactant function. Even when applied as an aerosol, surfactant might reach atelectatic lung regions due to its adsorption and lateral
spreading facilities. It is in line with these observations
that a significant improvement in gas exchange was noted in response to
surfactant nebulization, undertaken subsequent to the current protocol
of alveolar fibrin formation. The presently deposited surfactant quantity of 15 mg/kg body wt is well in line with previous
experimental surfactant replacement strategies employing aerosol
technology (12, 18, 20, 24, 25). However, the overall
efficacy of the transbronchial surfactant administration was somewhat
limited in the present study in fibrin-loaded lungs compared with that in lavage models (18, 20, 24). The most reasonable
explanation for this finding is the assumption that the presence of
fibrin interferes with the entrance of surfactant into the alveolar
spaces or that even strong apposition of alveolar walls ("fibrin
gluing") may occur.
To target alveolar fibrin formation more directly, nebulization of
urokinase was undertaken as a rescue regimen. When this approach was
tested in preceding pilot experiments, the pulmonary deposition
of ~10,000 U/kg body wt of uPA was found to significantly increase the Kfc, which may be due to the lysis
of extracellular matrix components, with subsequent enhanced fluid flux
under conditions of an increased pulmonary venous pressure challenge.
In contrast, pulmonary deposition of ~4,500 U/kg body wt of urokinase
did not exert any effect on lung barrier properties, ventilation
pressure, and pulmonary hemodynamics. This dose appears to be low
compared with the uPA doses currently used in acute myocardial
infarction, where 40,000-50,000 U/kg body wt may be employed
(26). It has to be kept in mind, however, that the volume
of the bronchoalveolar lining layer, to which the nebulized urokinase
is first distributed, is lower by many orders of magnitude than the
circulating blood volume, even under conditions of lung edema
formation. Moreover, measurement of the perfusate levels of urokinase
did not detect rapid passage of the aerosolized protease into the
intravascular space (ELISA technique; data not shown). The efficacy of
nebulized urokinase to cause fibrin lysis in the alveolar space is
documented by the increase in lavage fluid D-dimer levels in response
to this regimen. It is known from an in vitro study (8)
that a urokinase attack on surfactant-incorporating fibrin is capable of liberating functionally intact surfactant, which might then be
assumed to improve surface tension properties at the alveolar surface.
Indeed, some retardation of shunt flow development and perfusion of low
A/
areas was noted in the urokinase-treated lungs; however, the data did not significantly differ from the values
in nontreated lungs. Thus, although showing some efficacy, the
presently employed dose of urokinase, designed to avoid induction of
lung permeability increase by this protease, resulted only in a minor
improvement of gas exchange in the fibrin-loaded lungs.
Most interestingly, the combined aerosol administration of urokinase
and surfactant turned out to be the most effective approach for
improvement of A/
matching in the present
model of alveolar fibrin deposition. Notably, not only retardation of
shunt development but reversal of the extent of shunt flow was achieved
by this approach, with percentages of shunt at the end of the
experiments being in the same range as in the lungs undergoing
fibrinogen-alone administration (
7%). In addition, low numbers of
low
A/
areas were noted, thus resulting in a
markedly increased flow to lung units with normal
A/
ratios. Thus one very plausible
explanation might be that the surfactant facilitated the distribution
of urokinase to the fibrin-loaded alveolar spaces (surfactant as
carrier), thereby enhancing the overall fibrinolytic efficacy of the
nebulized uPA and converting most fibrin into fibrin split products;
the surfactant-inhibitory capacity of the split products only slightly surpasses that of fibrinogen. However, lavage fluid D-dimer levels were
not found to be increased in the urokinase-surfactant group compared
with the urokinase-alone group. Thus, as an alternative explanation,
some limited urokinase-induced fibrinolysis might allow better access
of the exogenously administered surfactant material to the alveolar
compartment, thereby enhancing the beneficial impact of the exogenous
surfactant material on surface tension regulation and alveolar stability.
In conclusion, the present study demonstrates that alveolar
fibrin formation, induced in an intact lung with otherwise
physiological function, provokes severe gas exchange abnormalities,
with a predominance of shunt flow. This finding is very compatible with
the hypothesis that fibrin formation in the alveolar compartment is a
potent inhibitor of surfactant function, resulting in a loss of
alveolar stability. Rescue administration of surfactant and urokinase, both aerosolized after formation of the fibrin deposits, reduced the
progressive development of shunt flow in this model, with surfactant
being more potent. The most efficient approach, however, was the
conebulization of surfactant and urokinase, which reversed shunt flow
and markedly increased perfusion of normal A/
areas. Clearly, it has to be kept in mind that these results were
obtained in an in vitro model, not under in vivo conditions of acute
respiratory failure. However, the proof of the concept (high efficacy
of conebulization of surfactant and urokinase) was beyond doubt
documented in this model. Thus combined surfactant and urokinase
administration may indeed offer a new therapeutic regimen in conditions
with marked and persisting alveolar fibrin formation such as severe or
delayed ARDS and pneumonia demanding mechanical ventilation (9,
16) and rapidly progressive idiopathic lung fibrosis (3,
17), in which each agent alone might not have easy access to the
fibrin-filled alveolar spaces. The currently employed dose of
urokinase was chosen to avoid induction of vascular leakage, but
further studies in intact animal models will be mandatory to figure out
whether coaerosolization of surfactant and a fibrinolytic agent may be operative without provoking lung bleeding. Beyond the present target
variable (restoration of gas exchange), this approach may also be of
interest in view of the hypothesis that delayed clearance of fibrin in
inflammatory lung diseases may favor the progression into lung
fibrosis, with surfactant-depleted and fibrin-glued alveolar spaces
serving as nidi for fibroblast invasion (concept of collapse
induration) (2).
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ACKNOWLEDGEMENTS |
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Our gratitude goes to Dr. P. D. Wagner (University of California, San Diego, La Jolla, CA) for supplying the computer program. We also thank R. L. Snipes (Department of Anatomy, Justus-Liebig-University Giessen, Giessen, Germany) for linguistically reviewing this manuscript.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 547.
Address for reprint requests and other correspondence: R. T. Schermuly, Zentrum für Innere Medizin, Justus-Liebig-Universität Giessen, Klinikstrasse 36, D-35392 Giessen, Germany (E-mail: ralph.schermuly{at}innere.med.uni-giessen.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 September 2000; accepted in final form 6 November 2000.
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