rhs-TM prevents ET-induced increase in pulmonary vascular permeability through protein C activation

Mitsuhiro Uchiba1, Kenji Okajima2, Kazunori Murakami2, Masayoshi Johno3, Mitsunobu Mohri4, Hiroaki Okabe2, and Kiyoshi Takatsuki1

Departments of 1 Medicine, 2 Laboratory Medicine, and 3 Dermatology, Kumamoto University School of Medicine, Kumamoto 860; and 4 Institute for Life Science Research Laboratory, Asahi Chemical Industry, Shizuoka 410-23, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation. In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C. rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12). Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM. ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM. However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C. These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.

acute respiratory distress syndrome; disseminated intravascular coagulation; leukocyte activation; cytokines; endothelial cell injury ; recombinant human soluble thrombomodulin; endotoxin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACUTE RESPIRATORY DISTRESS SYNDROME (ARDS) is one of the critical pathological events in sepsis that is associated with a mortality rate of >50% (4, 21). Cytokines and other inflammatory mediators derived from activated leukocytes have been implicated in the pathogenesis of ARDS (22, 29).

Thrombomodulin (TM) is an endothelial cell membrane glycoprotein that forms a high affinity complex with thrombin (20). TM plays an important role in the downregulation of coagulation (8). Thrombin bound to TM does not show procoagulant activity, such as conversion of fibrinogen into fibrin and the activation of factor V and VIII, but it can activate protein C to form activated protein C (APC) in plasma (8). APC thus formed exerts anticoagulant effects by inactivating factors Va and VIIIa, thereby regulating the coagulation cascade (23, 28).

TM also plays an important role in downregulation of lung injury. Recently, we demonstrated that recombinant human soluble (rhs) TM prevented endotoxin (ET)-induced pulmonary vascular injury by inhibiting leukocyte activation as well as the coagulation abnormalities in rats (24). Because APC has been implicated in the regulation of inflammatory reactions involving cytokines or activated leukocytes (7) and because we have previously demonstrated that APC prevented ET-induced pulmonary vascular injury by inhibiting leukocyte activation in rats (16), rhs-TM might inhibit the ET-induced pulmonary vascular injury by APC generation. However, because thrombin plays an important role in the pathophysiology of ARDS (10), it is possible that rhs-TM/thrombin binding may also contribute to the prevention of ET-induced pulmonary vascular injury in rats.

The present study was undertaken to investigate whether protein C activation by rhs-TM is responsible for prevention of ET-induced pulmonary vascular injury, as measured by inhibition of ET-induced leukocyte activation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. rhs-TM was kindly provided by Asahi Chemical Industry (Tokyo, Japan); the method used for preparation of rhs-TM has been described previously (9). Purified rhs-TM migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (9). This form of rhs-TM has plasma half-lives of 0.2 and 7 h in rats (2). Bovine serum albumin (BSA) was obtained from Sigma Chemical (St. Louis, MO), ET (lipopolysaccharide, Escherichia coli, serotype 055:B5) was from Difco (Detroit, MI), and Bolton-Hunter reagent and 51Cr were from Amersham International (Buckinghamshire, UK). A chromogenic substrate, S-2366 (Glu-Pro-Arg-p-nitroanilide), was obtained from Chromogenix (Stockholm, Sweden). All reagents used were of analytical grade.

Preparation of monoclonal antibodies against rhs-TM. Anti-rhs-TM monoclonal antibodies (MAbs) MF2, R5G12, and F2H5 were produced by the hybridoma technique in mice using rhs-TM as the antigen (12). In brief, the hybridoma supernatants obtained from BALB/c mice immunized with rhs-TM were assayed for antibody production. The selected hybridoma clones were expanded by intraperitoneal injection of hybridomas into BALB/c mice pretreated with pristane (Wako Chemical, Osaka, Japan). The ascites fluid was collected 1-2 wk after the injection. The immunoglobulin G antibodies were affinity purified from mouse ascites under sterile conditions on protein A Sepharose CL-4B.

Measurement of thrombin binding activity and protein C activation capacity of rhs-TM. Effects of various MAbs on thrombin binding to rhs-TM and protein C activation capacity of rhs-TM were determined according to a previously described method (1). Because fibrinogen clotting activity of thrombin is inhibited by rhs-TM/thrombin binding, thrombin-mediated fibrinogen clotting time was measured in the presence of rhs-TM and various MAbs to evaluate the thrombin binding activity by rhs-TM. Clotting time was measured at 37°C in a coagulometer (KC4A; Amelung, Lieme, Germany). rhs-TM (50 nM) was preincubated at 37°C for 2 min with 50 nM of MAb in 50 µl of a 25 mM tris(hydroxymethyl)aminomethane (Tris) · HCl buffer (pH 7.5) containing 0.15 M NaCl and 0.02% of Lubrol PX. After 50 µl of 3 mg/ml bovine fibrinogen in 25 mM Tris · HCl buffer (pH 7.5) containing 0.15 M NaCl were added to the preparation, 50 µl of 50 nM thrombin were added to initiate clotting.

Protein C activation by rhs-TM was evaluated as follows. rhs-TM (1 mM) was pretreated with an equimolar amount of various MAbs in 20 mM Tris · HCl (pH 7.5) containing 0.15 M NaCl, 5 mM CaCl2, and 0.5% BSA. After an incubation for 30 min at 37°C, thrombin and protein C were added at final concentrations of 1 mM and 0.15 µM, respectively. The reaction mixture was incubated at 37°C for 1 min. The reaction was terminated by the addition of 0.5 mg/ml argatroban. APC generation was measured spectrophotometrically (DU-64; Beckman, Irvine, CA) as the increase in absorbance at 405 nm after the addition of 0.5 mM of S-2366.

Treatment of rhs-TM with MAbs MF2, R5G12, and F2H5. rhs-TM was incubated at 37°C for 30 min with an equimolar concentration of MAb MF2, R5G12, or F2H5. Aliquots of the solution were assayed for protein C activation capacity and fibrinogen clotting activity, as described above, to determine the TM activity.

Animal model of ET-induced acute pulmonary vascular injury. The study protocol was approved by the Kumamoto University Animal Care and Use Committee, and the care and handling of the animals were in accordance with National Institutes of Health guidelines. Adult pathogen-free male Wistar rats (body mass 180-220 g; Nihon SLC, Hamamatsu, Japan) received an intravenous infusion of 125I-labeled BSA [specific activity, 3.0 × 1014 counts · min-1 (cpm) · mol-1] prepared with Bolton-Hunter reagent (2.0 × 105 cpm/kg body wt) 5 min before an intravenous bolus dose of 5 mg/kg ET was injected into the tail vein as described previously (16). rhs-TM (1 mg/kg) treated with or without MAbs against rhs-TM was administered intravenously 30 min before injection of ET. Control animals received saline only. Animals were anesthetized by an intraperitoneal injection of 50 mg/kg pentobarbital sodium and were exsanguinated via the abdominal aorta 6 h after administration of ET. Blood samples were collected in tubes containing a 0.1 volume of 3.8% sodium citrate. Blood was centrifuged at 2,000 g for 10 min. The lung vasculature was perfused through the right cardiac ventricle with 10 ml of saline. The lungs were then removed and weighed, and the amount of radioactivity remaining within the tissue was measured with a gamma scintillation counter (model 5130; Packard Instrument, Downers Grove, IL). Pulmonary vascular damage induced by ET was evaluated by increased vascular permeability as expressed by a permeability index. The permeability index is defined as the distribution ratio of 125I-BSA between lung and blood, that is, the ratio of the amount of radioactivity present within lung tissue to the amount of radioactivity present in 1 ml of blood obtained at the time of death, as described previously (16). The change in pulmonary vascular permeability evaluated by this method was well correlated with the change in wet-to-dry weight ratio of rat lungs after ET challenge (27).

To assess lung hemorrhage or pulmonary congestion induced by the administration of ET, we measured the accumulation of 51Cr-labeled red blood cells (RBC) in rats receiving ET and in controls. RBC were labeled with 51Cr as previously described (24). Animals were injected intravenously with 51Cr-labeled RBC (50 µl containing 8.0 × 104 cpm) 30 min before injection of ET. Rats were killed 6 h later, and the radioactivity in the lungs was measured and compared with that present in blood. No significant increase in 51Cr-labeled RBC was observed 6 h after ET administration in perfused or nonperfused lung (data not shown). We thus excluded ET-induced lung hemorrhage or significant pulmonary blood pooling in this animal model.

Bronchoalveolar lavage. Bronchoalveolar lavage (BAL) was performed as described previously (24). In brief, after intraperitoneal administration of pentobarbital sodium, an incision was made in the anterior neck, and a catheter was secured in the trachea with a surgical suture. BAL was performed with 5 ml of saline. Radioactivity in the BAL fluid was measured with a gamma scintillation counter.

Measurement of lung myeloperoxidase activity. Ninety minutes after ET administration, 10 ml of saline were perfused through the lung vasculature into the right cardiac ventricle. The lungs were then removed, and accumulation of leukocytes was assessed by measuring myeloperoxidase (MPO) activity in rat lungs according to a previously described method (16). In brief, lung samples were homogenized using 6 ml of homogenizing buffer containing 0.1 M phosphate buffer (pH 6.0) and 1% hexadecyltrimethylammonium bromide. The homogenate was then sonicated and was centrifuged at 4,500 g for 30 min at 4°C. The supernatant was measured for MPO activity as follows. The 0.1-ml test sample was mixed with 0.6 ml of 0.05 M phosphate buffer containing 0.167 mg/ml o-dianisidine dihydrochloride and 0.0005% H2O2 (pH 6.0). The change in absorbance at 460 nm was measured over 1 min at 25°C in a spectrophotometer (DU-54; Beckman). One unit of enzyme activity was defined as the amount of MPO present that causes a change in absorbance of 1.0 per minute at 460 nm and was expressed as units per gram of lung weight.

Histopathological studies of the lungs. Histopathological examination of the lungs was performed 6 h after administration of 5 mg/kg ET. Samples were fixed with 10% Formalin, embedded in paraffin, sectioned into 6-µm pieces, and stained with hematoxylin and eosin. Samples were analyzed by a pathologist blinded to the animal's group assignment. Ten randomly selected fields per slide were read under oil at ×1,000 magnification by an observer who did not know the animal's group assignment. Fields containing large vessels or bronchi were excluded. The number of polymorphonuclear neutrophils per field was counted and normalized to alveoli per field to control for inflation of the lung.

Assays. The plasma concentration of fibrinogen was determined 6 h after ET administration as the amount of coagulable protein, according to the method of Clauss (6). Fibrin and fibrinogen degradation products (E) [FDP(E)] were measured 6 h after ET administration in rat serum samples with the latex agglutination assay, as previously described (16).

Statistical analysis. Data are presented means ± SD. Data were analyzed by analysis of variance and Scheffé's post hoc test. P < 0.05 was accepted as statistically significant.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effects of MAbs on thrombin binding activity and protein C activation capacity of rhs-TM. MAb F2H5 almost completely inhibited thrombin binding to rhs-TM, but MAbs R5G12 and MF2 did not inhibit thrombin binding to rhs-TM (Fig. 1). The protein C activation capacity of rhs-TM was almost completely inhibited by MAb F2H5 and was inhibited by MAb R5G12 by ~66% but was not inhibited by MAb MF2 (Fig. 2). Thus MAb F2H5 inhibits thrombin binding and subsequent protein C activation by rhs-TM, and MAb R5G12 inhibits protein C activation by rhs-TM. MAb MF2 does not affect thrombin binding to rhs-TM or subsequent protein C activation. These activity experiments were repeated using the solutions that were administered to rats for testing (data not shown). These solutions comprised rhs-TM, various MAbs, and saline.


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Fig. 1.   Effects of recombinant human soluble (rhs) thrombomodulin (TM) and monoclonal antibody (MAb)-treated rhs-TM on thrombin binding to rhs-TM. Effects of rhs-TM and MAb (F2H5, R5G12, and MF2)-treated rhs-TM on thrombin-mediated fibrinogen clotting time were determined. Incubation of rhs-TM with various MAb and the measurement of clotting time are described in MATERIALS AND METHODS. Saline was added instead of rhs-TM or MAb-treated rhs-TM in the control experiment. Data are expressed as means ± SD of triplicate experiments. * P < 0.01 vs. control; ** P < 0.01 vs. rhs-TM.


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Fig. 2.   Effect of rhs-TM and MAb-treated rhs-TM on the ability of rhs-TM to activate protein C. Protein C activation (APC) capacities of rhs-TM and MAb (F2H5, R5G12, and MF2)-treated rhs-TM were measured according to the method as described in MATERIALS AND METHODS. Generation of activated protein C by rhs-TM was determined by using a chromogenic substrate, S-2366, as described in MATERIALS AND METHODS. Saline was added instead of MAb in the control experiment. Data are expressed as means ± SD of triplicate experiments. * P < 0.01 vs. control.

Effects of MAb-treated rhs-TM on ET-induced pulmonary accumulation of leukocytes, increase in pulmonary vascular permeability, and intravascular coagulation in rats. In this animal model of septicemia, pulmonary accumulation of leukocytes, as evaluated by determining lung MPO activity, increases with time and peaks at 90 min after ET administration. Pulmonary vascular permeability to 125I-BSA increases with time after administration of ET and peaks at 6 h of ET administration (16). Intravenous administration of 1 mg/kg rhs-TM significantly inhibits (1) the increase in pulmonary accumulation of leukocytes 90 min after ET administration and (2) the increase in pulmonary vascular permeability 6 h after ET administration (16). rhs-TM (1 mg/kg) also attenuates intravascular coagulation induced by ET at 6 h (24). There is no significant increase in the ratio of 125I-BSA concentration in the BAL fluid to that in blood 6 h after administration of ET.

To determine whether rhs-TM might inhibit the increase in pulmonary vascular permeability by binding thrombin or by activation of protein C, we examined the effect of MAb-treated rhs-TM on the ET-induced pulmonary accumulation of leukocytes and the subsequent increase in pulmonary vascular permeability. Pretreatment of rhs-TM with MAbs F2H5 or R5G12 did not prevent the ET-induced pulmonary accumulation of leukocytes or the increase in pulmonary vascular permeability observed at 90 min and 6 h after ET administration, respectively (Fig. 3, A and B). Pretreatment of rhs-TM with the MF2 MAb prevented both the pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability (Fig. 3, A and B). As stated earlier, MAb MF2-treated rhs-TM can activate protein C and can bind thrombin.


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Fig. 3.   Effects of rhs-TM and MAb-treated rhs-TM on endotoxin (ET)-induced pulmonary accumulation of leukocytes (A), pulmonary vascular injury (B), decrease in the plasma concentration of fibrinogen (D), and increase in the serum concentration of fibrin and fibrinogen degradation products [FDP(E)] (C) in rats. rhs-TM (1 mg/kg) and MAb (F2H5, R5G12, and MF2)-treated rhs-TM were administered intravenously 30 min before intravenous injection of 5 mg/kg ET. Pulmonary accumulation of leukocytes was assessed by measuring lung myeloperoxidase (MPO) activity 90 min after ET administration (A). The pulmonary vascular permeability index was determined 6 h after ET administration (B), and plasma concentrations of fibrinogen (D) and serum concentrations of FDP(E) (C) were determined 6 h after ET administration. Control animals received 0.9% NaCl instead of ET. Data are expressed as means ± SD; n, no. of animals in experiments. * P < 0.01 vs. control; dagger  P < 0.01 vs. ET.

The plasma level of fibrinogen began to decrease 2 h after ET administration, reaching its minimum at 6 h after ET administration (16). The serum concentration of FDP(E) increased significantly after administration of ET, peaking at 6 h (25). Pretreatment of rhs-TM with the F2H5 MAb did not prevent the ET-induced intravascular coagulation, as evidenced by the reduction of plasma fibrinogen level and increase in serum FDP(E) level observed 6 h after ET administration (Fig. 3, C and D). In contrast, rhs-TM pretreated with the MAb R5G12 or the MAb MF2 prevented the ET-induced intravascular coagulation induced by ET (Fig. 3, C and D).

Effects of rhs-TM and rhs-TM pretreated with MAb on changes in the pulmonary histological findings induced by ET. Microscopic observation of lung tissue 6 h after administration of ET revealed edematous changes and infiltration of neutrophils in the interstitial space (Fig. 4B). These changes were not present in lung tissue from animals treated with saline alone (Fig. 4A). Administration of rhs-TM markedly reduced interstitial edema and neutrophilic infiltration (Fig. 4C). Administration of MAb MF2-treated rhs-TM also reduced interstitial edema and neutrophilic infiltration (Fig. 4F). However, administration of MAb R5G12-treated rhs-TM or F2H5-treated rhs-TM did not reduce ET-induced histological changes in the lung (Fig. 4, D and E).


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Fig. 4.   Microscopic observation of lung tissue from animals treated with saline (A, ×400), ET (B, ×400), ET plus rhs-TM (C, ×400), ET plus MAb F2H5-treated rhs-TM (D, ×400), ET plus MAb R5G12-treated rhs-TM (E, ×400), and ET plus MAb MF2-treated rhs-TM (F, ×400). Histopathological examination of the lungs was performed 6 h after 5 mg/kg ET administration. Saline or 1 mg/kg rhs-TM was administered intravenously 30 min before injection of ET. Interstitial edema formation and infiltration of neutrophils were observed in ET-treated animals (B), whereas these changes were not observed in saline-treated animals (A). rhs-TM markedly reduced interstitial edema and neutrophilic infiltration (C). MAb F2H5-treated rhs-TM and MAb R5G12-treated rhs-TM did not reduce the ET-induced histological changes (D and E). MAb MF2-treated rhs-TM markedly reduced interstitial edema and neutrophilic infiltration (F).

Examination of fixed lung tissue by light microscopy revealed differing numbers of neutrophils per alveolus in the lung tissue 6 h after ET administration compared with that of control lung tissue (Table 1). Administration of rhs-TM markedly reduced neutrophilic infiltration induced by ET. Administration of MAb MF2-treated rhs-TM reduced neutrophilic infiltration induced by ET, whereas administration of MAb R5G12-treated rhs-TM or F2H5-treated rhs-TM did not (Table 1).

                              
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Table 1.   Neutrophils in lung tissue from rats

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

In the present study, the anti-inflammatory activity of rhs-TM was correlated with the protein C activating ability of rhs-TM. In particular, rhs-TM did not inhibit the ET-induced increase in pulmonary vascular permeability when the protein C activation ability of rhs-TM was selectively inhibited by pretreatment of rhs-TM with MAb R5G12. This suggests that inhibition of the ET-induced increase in pulmonary vascular permeability by rhs-TM may be dependent on the ability of rhs-TM to activate protein C but is not dependent on its ability to bind thrombin, although rhs-TM pretreated with MAb F2H5 failed to prevent the ET-induced increase in pulmonary vascular permeability. The observations in the present study are consistent with our previous report demonstrating that APC prevents the ET-induced increase in pulmonary vascular permeability (16). In addition, we have recently demonstrated that APC may prevent the ET-induced increase in pulmonary vascular permeability by inhibiting tumor necrosis factor-alpha (TNF-alpha ) production by monocytes in rats (17). TNF-alpha activates neutrophils to release the various inflammatory mediators capable of damaging endothelial cells (1, 13). Thus rhs-TM may inhibit the ET-induced increase in pulmonary vascular permeability through generation of APC, which can inhibit leukocyte activation.

The fact that R5G12-treated rhs-TM did not inhibit ET-induced pulmonary vascular permeability suggests that procoagulant activity of thrombin may not be involved in the pathological process leading to this permeability in the animal septicemia model used in this study. However, coagulation abnormalities have been considered to be an important predisposing factor for ARDS (4, 15). The fibrin and fibrin degradation products present in the alveolar spaces have been shown to be chemoattractants for leukocytes that play an important role in the pathogenesis of ARDS (3, 11). Because we did not observe fibrin deposition in the alveolar spaces and because the amount of 125I-BSA intravenously injected was not increased in BAL in rats given ET, ET-induced lung injury might be limited to the endothelial cells in the animal model of septicemia used in the present study. This may explain why inhibition of intravascular coagulation did not attenuate the ET-induced increase in pulmonary vascular permeability in the present study. However, prevention of intravascular coagulation by rhs-TM, which is mediated mainly by binding thrombin, may contribute to the attenuation of ARDS in a clinical setting.

One may speculate that the pretreatment of rhs-TM with any MAb adversely affects the ability of rhs-TM to prevent the ET-induced increase in pulmonary vascular permeability. However, this seems unlikely because treatment of rhs-TM with MAb MF2, which affects neither thrombin binding nor protein C activation by rhs-TM, did not affect the ability of rhs-TM to prevent the ET-induced increase in pulmonary vascular permeability.

We have previously reported that antithrombin III (ATIII) prevents ET-induced pulmonary vascular injury by inhibiting leukocyte activation by stimulating endothelial release of prostacyclin (25). For the stimulation of prostacyclin release from endothelial cells, interaction of ATIII with glycosaminoglycans on endothelial cells is important (26). These observations, together with the present findings, suggest that both the ATIII-glycosaminoglycan system and the TM-protein C system, two major important anticoagulant mechanisms operating on the endothelial cell surface, may regulate the leukocyte activation as well as the coagulation system. Thus these two physiological anticoagulants may be useful for prevention of intravascular coagulation and ARDS in patients with septicemia in which leukocytes play an important role (18, 19).

Whereas the present studies were carried out with soluble TM, this protein is an endothelial cell surface protein. TM is highly enriched in the pulmonary vascular lining (14). Because TM binds thrombin to activate protein C in the pulmonary microcirculation, it is possible that TM may play an important role not only in modulation of coagulation but also in prevention of pulmonary endothelial cell injury by regulation of the activation of leukocytes. Further studies should be done to evaluate such a novel role of endothelial TM in the pulmonary microcirculation.

    FOOTNOTES

Address for reprint requests: K. Okajima, Dept. of Laboratory Medicine, Kumamoto Univ. School of Medicine, Honjo 1-1-1, Kumamoto 860, Japan.

Received 14 February 1997; accepted in final form 8 July 1997.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Lung Cell Mol Physiol 273(4):L889-L894
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