INTRODUCTION
Pseudomonas aeruginosa is a common Gram-negative nosocomial pathogen associated with respiratory tract infections (1). Critically ill patients are susceptible to P. aeruginosa infections, because airway colonization frequently occurs, at least in part due to the widespread use of broad-spectrum antibiotics in the intensive care unit (2). Ventilator-associated pneumonia with P. aeruginosa has a high morbidity and mortality compared with pneumonia caused by other pathogens (3). Pulmonary activation of coagulation, frequently referred to as pulmonary coagulopathy, is a hallmark of pneumonia. Inflammation and consequent activation of coagulation and inhibition of fibrinolysis result in excessive fibrin deposition within the airways, compromising pulmonary integrity and function (4). The local changes in alveolar coagulation and fibrinolysis closely resemble the systemic changes found in sepsis.
In patients with severe sepsis or septic shock, systemic treatment with recombinant human activated protein C (rh-APC), one of the natural anticoagulants, decreased morbidity and mortality and led to more rapid resolution of respiratory failure, as compared with placebo (5). Also, patients with pneumonia as the source of sepsis seemed to benefit most from rh-APC treatment (6). Several preclinical studies have evaluated the effect of rh-APC on lung injury: in rodents, intratracheal (7) and systemic (8) application of rh-APC attenuated bleomycin-induced and endotoxin-induced lung injury, respectively. In a recent clinical study, systemic rh-APC treatment significantly reduced pulmonary dead space fraction in patients with acute lung injury (ALI) but neither reduced the duration of mechanical ventilation nor improved overall outcome (9).
Antithrombin (AT), another natural anticoagulant, is rapidly consumed during inflammatory states. Lung-protective effects of AT have been demonstrated in a limited number of patients with sepsis (10). Antithrombin may exert lung-protective effects through increased prostacyclin-mediated inhibition of cytokines and inhibition of leukocyte activation and migration (11, 12). It is also hypothesized that AT competes with bacterial toxins for binding on endothelial cell proteoglycans, thereby limiting the inflammatory response after bacterial challenge (13).
Heparin is a potent activator of AT, but in a recent clinical trial, continuous infusion of low-dose unfractioned heparin did not affect mortality in patients with sepsis, nor was mortality affected in a subgroup of patients with pneumonia (14). However, no subgroup analysis was performed on patients with respiratory failure or ALI/acute respiratory distress syndrome. Heparin successfully prevented fibrin deposition and attenuated lung injury in ovine and porcine models of ALI (15, 16). Danaparoid exerts its anticoagulant effects primarily by inhibiting factor Xa. Systemically administered danaparoid has been shown to prevent endotoxin-induced ALI in rats (17). Because danaparoid has a longer pharmaceutical half-life than heparin, danaparoid may be better suited than heparin to maintain constant and sufficient (pulmonary) levels to exert its anticoagulant and anti-inflammatory effects.
An important drawback for the systemic administration of anticoagulants is the increased risk of bleeding complications. Local administration may prevent this risk, and in previous studies, we demonstrated attenuation of pulmonary procoagulant changes and limitation of lung injury (18, 19). However, it remains unclear which anticoagulant compound most adequately exerts intrapulmonary activity with a minimum of systemic effects. Therefore, we investigated the intrapulmonary and systemic effects of nebulization of the aforementioned anticoagulants in a rat model of P. aeruginosa pneumonia.
MATERIALS AND METHODS
The Institutional Animal Care and Use Committee of the Academic Medical Center approved all experiments. All animals were handled in accordance with the guidelines prescribed by Dutch legislation and the international guidelines on protection, care, and handling of laboratory animals.
Induction of pneumonia
Pneumonia was induced in male Sprague-Dawley rats (250-300 g) (Harlan, Horst, the Netherlands) by intratracheal instillation of 108 colony-forming units of P. aeruginosa (PAO1, in a total volume of 250 μL of bacterial suspension), which was cultured as previously described (20).
Interventions
Rats were randomized to local treatment with placebo (0.9% saline) or local treatment with 5,000 μg/kg rh-APC (drotrecogin alfa activated; Eli Lilly, Indianapolis, Ind), 500 U/kg plasma-derived AT (plasma-derived AT III; Baxter, Vienna, Austria), 1,000 IU/kg heparin (Leo Pharmaceutical, Ballerup, Denmark), or 250 anti-Xa U/kg danaparoid (Orgaran; Organon, Oss, the Netherlands), all by means of nebulization (n = 7 per group). Healthy animals without pneumonia served as controls (n = 4). Dosages and timing of anticoagulant medication were determined using data from previous studies (21-23). The first administration of each agent was 30 min before intratracheal challenge with P. aeruginosa. Nebulization of saline, rh-APC (elimination half-life, 45 min), heparin (elimination half-life, approximately 1½ h), and danaparoid (elimination half-life, 25 h) was repeated every 6 h until rats were killed. Considering its longer elimination half-life (19-72 h), AT was administered only once.
For local treatment, we used an adapted dynamic airflow, nose-only exposure system, which allows direct exposure of nebulized agents to the noses of the animals. This system consisted of a concentric manifold connected to the necks of bottle-like restraint tubes (CHT 249 restraint tube; CH technologies Inc, Westwood, NJ) in which the animals were confined with their noses adjacent to the bottlenecks. The bottles were detachable, allowing disassembly of the device for cleaning. The inhalation chamber was suitable to accommodate several rats at once. One extra outlet was available for measuring the pressure and atmosphere sampling inside the inhalation chamber. The aerosolized agent was supplied to the upper end of the manifold, flowing adjacent to the noses of the individual animals, and then was drawn out through the bottom of the manifold. The aerosol atmosphere was generated using the Aeroneb Pro Nebulizer (Aerogen Ltd, Galway, Ireland). The Aeroneb Pro Nebulizer uses a vibrating mesh with multiple apertures to generate a fine-particle, low-velocity aerosol and produces aerosols with an average size of 2.1 μm. The aerosols were directed to the inhalation chamber by a constant oxygen flow (2 L/min).
Sampling
At 16 h after inoculation, rats were killed with intraperitoneal injections of ketamine (80 mg/kg; Eurovet, Bladel, the Netherlands) and medetomidine (0.5 mg/kg; Novartis, Arnhem, the Netherlands). Blood (5 mL) was collected from the inferior vena cava in citrated (0.109 M) vacutainer tubes (Becton Dickinson, Breda, the Netherlands). The right lung was ligated, and the left lung was lavaged three times with 2 mL of ice-cold sterile saline (average total recovery, 4.5-5 mL). The right superior lobe was fixed in 10% buffered formalin and embedded in paraffin. The remaining lung lobes were weighed and homogenized in four volumes of sterile saline (i.e., 4× lung weight [in milligrams] in microliters) using a tissue homogenizer (Biospec Products, Bartlesville, Okla).
Measurements
Total cell numbers in lavage samples were determined in a Coulter counter (Coulter Electronics, Hialeah, Fla). Neutrophil counts in lavage fluids were performed on cytospin preparations stained with Giemsa. For bacterial quantification in lungs and blood, serial 10-fold dilutions of lavage fluid, lung homogenates, and whole blood were made in sterile isotonic saline and plated onto sheep-blood agar-coated plates, incubated at 37°C in 5% CO2, and counted after 20 h. For coagulation assays, cell-free supernatants from lavage fluid and blood were used. For cytokine and chemokine measurements in lung homogenates, cell-free supernatants were used from lung homogenates that were diluted 1:1 in lysis buffer (150 nmol/L NaCl; 15 mmol/L Tris; 1 mmol/L MgCl2-H2O; 1 mmol/L CaCl2; 1% Triton X-100; and 100 μg/mL pepstatin A, leupeptin, and aprotinin).
Thrombin-AT complexes (TATc; Behring, Marburg, Germany) and fibrin degradation products (FDPs; Asserachrom D-Di; Diagnostica Stago, Asnières-sur-Seine, France) were measured in bronchoalveolar lavage fluid (BALF) using enzyme-linked immunosorbent assay. Antithrombin, plasminogen activator activity (PAA), and plasminogen activator inhibitor 1 (PAI-1) activity were measured by automated amidolytic assays. The TATc-to-AT ratio was calculated to describe thrombin activation in excess of AT changes. Levels of TNF-α, IL-6, and cytokine-induced neutrophil chemoattractant 3 (CINC-3) (R&D Systems, Abingdon, United Kingdom) and myeloperoxidase (MPO) (HyCult Biotechnology b.v., Uden, the Netherlands) were measured using enzyme-linked immunosorbent assay in lung homogenates. Total protein was determined with a bicinchoninic acid protein assay kit (Pierce, Rockford, Ill).
Lung histopathology
Histopathology was analyzed and scored by two investigators who were blinded for group identity. Four-micrometer sections were stained with hematoxylin and eosin. To score lung inflammation and damage, the entire slide surface was analyzed with respect to the following variables: alveolar inflammation, interstitial inflammation, endothelialitis, bronchitis, edema, pleuritis, and thrombus formation, as previously described (20). Each variable was scored on a scale from 0 to 4 (0, absent; 1, mild; 2, moderate; 3, severe; 4, very severe). The total histopathology score was expressed as the sum of scores for all variables.
Statistical analysis
All data are expressed as mean (SD) or median with interquartile ranges, where appropriate. Comparisons between the experimental groups and the saline-treated placebo group were performed using one-way ANOVA or Kruskal-Wallis test, followed by post hoc Dunnett or Dunn test, depending on data distribution. P < 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 16.0 (SPSS, Chicago, Ill) and Prism 4.0 (GraphPad Software, San Diego, Calif).
RESULTS
Pneumonia
Typical clinical symptoms of illness (pilo erection, decreased activity, arched back, decreased food and water intake, and increased respiratory rate) occurred shortly after instillation of P. aeruginosa. On average, the animals lost 8% of their bodyweight over 16 h, presumably mainly due to dehydration. Two rats (one in the placebo group and one in the plasma-derived AT group) died shortly after intratracheal manipulation because of laryngeal edema caused by the procedure. Rats killed 16 h after the bacterial challenge had evident diffuse bilateral macroscopic lung abnormalities.
Pulmonary coagulation and fibrinolysis
Pseudomonas aeruginosa pneumonia was associated with local activation of coagulation and inhibition of fibrinolytic activity (Fig. 1). Indeed, pulmonary levels of TATc and FDPs were increased, whereas levels of AT were decreased after induction of pneumonia. Levels of PAA were decreased, which was, at least in part, the result of increased levels of PAI-1, the main inhibitor of fibrinolytic activity in the lungs.
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Fig. 1:
The effects of nebulized anticoagulants on pulmonary levels of TATc, AT activity (AT), TATc-to-AT ratio, PAA, PAI-1, and FDPs, 16 h after intratracheal bacterial challenge (P. aeruginosa, PAO1, 108 colony-forming units). AT indicates plasma-derived human AT; Hep, heparin; Dan, danaparoid. Data represent mean (SD). *P < 0.05; **P < 0.01 vs. placebo.
Local treatment with rh-APC significantly attenuated the rise in TATc and PAI-1 levels; in addition, AT activity was largely preserved, and FDP generation limited. Administration of plasma-derived AT resulted in supranormal levels of AT activity. Local treatment with AT resulted in significant attenuation of TATc production and limited FDP generation. Plasminogen activator activity levels were not affected.
The ratio of TATc to AT in BALF was markedly elevated in the placebo-treated animals. The elevation, however, was attenuated by all anticoagulants, yet most pronounced by rh-APC and AT.
Systemic coagulation and fibrinolysis
Pseudomonas aeruginosa pneumonia was accompanied by systemic activation of coagulation and inhibition of fibrinolysis, as reflected by increased plasma levels of TATc and decreased levels of PAA, respectively (Fig. 2). Local treatment with rh-APC reduced systemic TATc levels, whereas local administration of plasma-derived AT increased systemic TATc levels. The ratios of intrapulmonary to systemic TATc concentrations in both the rh-APC- and the plasma-derived AT-treated animals closely resembled those in the control group (Fig. 3). Heparin and danaparoid did not affect systemic coagulation. None of the nebulized anticoagulant treatments altered plasma PAA.
Fig. 2:
The effects of nebulized anticoagulants on systemic levels of TATc and systemic PAA, 16 h after intratracheal bacterial challenge. Data represent mean (SD). *P < 0.05, **P < 0.01 vs. placebo.
Fig. 3:
The ratios of intrapulmonary to systemic concentrations of TATc and PAI, 16 h after intratracheal bacterial challenge. Data represent mean(SD). **P < 0.01 vs. placebo.
Bacterial clearance from lungs
Bacterial loads of P. aeruginosa in lungs were similar in all groups at 16 h after inoculation. Bacteremia was not observed in any of the rats (Fig. 4).
Fig. 4:
The effects of anticoagulants on numbers of P. aeruginosa colony-forming units in BALF, 16 h after intratracheal bacterial challenge. Data represent median (IQR).
Inflammatory responses
There were no differences in wet lung weights between study groups (data not shown). However, there was an evident increase in the total cell number in the lungs during P. aeruginosa pneumonia, which was mostly attributed to neutrophil influx (Table 1). The relative or absolute numbers of neutrophils in lavage fluid were not affected by any treatment, nor was MPO activity or total protein levels altered. Pulmonary levels of TNF-α, IL-6, and CINC-3 were highly variable (Fig. 5). There were no differences in pulmonary levels of TNF-α, IL-6, and CINC-3 between treatment groups.
Table 1:
Total cell and neutrophil counts in BALF
Fig. 5:
TNF-α, IL-6, and CINC-3, determined in lung homogenates 40 h after intratracheal bacterial challenge. Dotted lines stipulate the normal values in healthy animals and untreated animals with pneumonia. Data represent median (IQR).
Histopathology
At 16 h after induction of P. aeruginosa pneumonia, pulmonary histology showed inflammatory infiltrates in all rats. Interstitial inflammation, endothelialitis, bronchitis, and edema were present to a variable extent. There were no differences in the total histopathology scores between treatment groups (Fig. 6).
Fig. 6:
Histopathologic changes in P. aeruginosa pneumonia. Shown are representative photomicrographs of hematoxylin and eosin-stained paraffin sections of lung tissue from rats treated with (A) placebo, (B) recombinant human APC, (C) plasma-derived AT, (D) heparin (Hep), and (E) danaparoid (Dan), at 16 h after bacterial challenge (original magnification ×100). F, Total histopathology scores are presented as median with interquartile range.
DISCUSSION
In this study, we demonstrate that, in a rat model of P. aeruginosa pneumonia, nebulization of rh-APC or plasma-derived AT potently inhibits bronchoalveolar activation of the coagulation system. Both rh-APC and plasma-derived AT affect systemic coagulation as reflected by a change in plasma levels of TATc. Heparin and danaparoid do not alter pulmonary fibrin turnover in this model. Finally, there are no changes in pulmonary inflammation or bacterial clearance from the respiratory tract, despite clear (local) anticoagulant effects of rh-APC and plasma-derived AT in the lungs.
Coagulation and inflammation are cardinal host defense mechanisms, mutually dependent in mounting an adequate immune response against potentially injurious stimuli (24). Interference with either the inflammatory or coagulation response may impede the primary host defense mechanisms. Previous animal studies showed that intervening in the initial procoagulant response could be detrimental, as formation of thrombi is thought to limit hematogenic bacterial spread. In a murine and rat model of P. aeruginosa-induced pneumonia, systemically administered rh-APC or AT led to increased pulmonary capillary permeability, enhanced TATc formation, and increased levels of TNF-α and IL-6 in serum and BALF (25, 26). However, there is a substantial body of evidence, suggesting favorable effects (27). Systemic administration of rh-APC improves pulmonary function and attenuates lung injury in ovine models of smoke- or oleic acid-induced lung injury (28, 29). Comparable effects were demonstrated with systemic rh-APC administration in Pseudomonas- or endotoxin-induced lung injury in rats and sheep, respectively (20, 30). In previous studies with Streptococcus pneumoniae and LPS-induced lung injury, we found significant reductions in coagulation activation in the pulmonary compartment as expressed by attenuated levels of TATc and FDPs (18, 19). In our current study, pulmonary coagulation activation was attenuated as assessed by the levels of TATc and FDPs. Consequently the level of AT was largely preserved. The profibrinolytic effect of rh-APC was reflected in the suppressed level of PAI-1, although this did not result in enhanced PAA.
As opposed to potential detrimental effects (25, 26), inhibition of pulmonary coagulation activation and limitation of lung injury were found when AT was systemically administered in rats with S. pneumoniae pneumonia (22). In our own model of S. pneumoniae-induced pneumonia, nebulization of plasma-derived AT led to histopathologic reduction of lung injury and a decrease of bacterial outgrowth (18).
In our study, the systemic effects on coagulation suggest that the nebulized anticoagulants are leaking from the alveolar compartment into the systemic circulation. Recombinant human APC attenuated systemic TATc levels, whereas plasma-derived AT, paradoxically, caused plasma TATc to increase. This increase in plasma TATc is probably due to the time point investigated. In this model of P. aeruginosa pneumonia, activation of coagulation leads to the formation of thrombin. Administration of AT will bind to thrombin, leading to an initial rise in TATc (25). Thereafter, thrombin generation is attenuated through inhibition of factors IXa, Xa, XIa, and XIIa and tissue factor VIIa complex by AT, resulting in a reduction of TATc (31). The ratio of pulmonary to systemic coagulation activation, as expressed by TATc, closely resembles the physiological state after administration of rh-APC and plasma-derived AT, suggesting normalization of the P. aeruginosa-induced procoagulant state, by these agents.
Heparin and danaparoid affected pulmonary coagulation as expressed by the TATc-to-AT ratio, but did not affect systemic coagulation. Underdosing may have been an issue; however, the same dosages did affect coagulation in endotoxin-induced lung injury in rats (32). Another explanation could be the low levels of pulmonary AT. The major anticoagulant effects of heparin and danaparoid are exerted via AT, and it may well be that AT consumption (as seen in our study, Fig. 2) leads to impaired actions of heparin and danaparoid. Previous animal studies have demonstrated that combined AT with heparin or danaparoid effectively reduces ALI (32).
One single-center phase 1 trial, in which 16 mechanically ventilated patients with ALI received nebulized heparin, showed this strategy to result in minimally prolonged systemic clotting times. Because of the design, no conclusions could be drawn regarding clinical effects; however, no adverse events were reported, and nebulization was deemed feasible and safe in patients with ALI (33). In a post hoc analysis, significantly reduced levels of TATc and FDPs were demonstrated in BALF of the patients who received 400,000 U/d, indicating amelioration of coagulation activation (34). In a recent study of critically ill patients expected to require prolonged mechanical ventilation, nebulization of heparin was associated with more ventilator-free days, regardless of the presence of ALI at the time of enrollment (35).
Notably, hematogenic dissemination of infection is suggested as a potential drawback of anticoagulant therapy in pneumonia (25, 26). However, bacteremia did not occur in any of the rats in our study.
There are several limitations to our study. First, this is an animal model of pneumonia and consequently a simplified model of a complex and heterogeneous patient population. We have used a pretreatment model, useful when exploring novel approaches and mechanisms; however posttreatment models more closely resemble the clinical situation. Furthermore, no antibiotics were administered. The chosen dosages for each anticoagulant agent are determined based on data from previous studies and pilot studies, combined with the efficacy of our nose-only exposure system, and possibility of each agent to dissolve to an acceptable volume for nebulization. For rh-APC and plasma-derived AT, this approach led to a significant attenuation of pulmonary coagulopathy; however, lower dosages may also have been sufficient. At the same time, the dosages of heparin and danaparoid may not have been sufficient to affect pulmonary coagulation. To achieve anti-inflammatory effects, higher dosages of these agents may be required than during anticoagulant use. Moreover, aerosol characteristics of the drugs we studied probably have been different. The fact that the animals were treated intermittently may be another limitation to our study, because continuous administration may have been more effective for heparin and danaparoid. Bacteremia was not observed, but the observational period was only 16 h, and this model was not designed to evaluate its occurrence.
In conclusion, pulmonary coagulopathy as a result of pneumonia may allow for interventions aiming at local administration of anticoagulants, thereby reducing the potential harmful adverse effects of systemic administration. Although rh-APC and plasma-derived AT did not alter bacterial clearance or affect the inflammatory response in rats challenged with P. aeruginosa, they affected pulmonary coagulation without inducing profound systemic effects, making them promising agents for local therapy.
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