AJP - Lung Fuel your research with LabChart
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 289: L946-L953, 2005. First published July 8, 2005; doi:10.1152/ajplung.00188.2004
1040-0605/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/6/L946    most recent
00188.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sawafuji, M.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawafuji, M.
Right arrow Articles by Kobayashi, K.

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Makoto Sawafuji,1 Akitoshi Ishizaka,2 Mitsutomo Kohno,1 Hidefumi Koh,2 Sadatomo Tasaka,2 Yoshiki Ishii,3 and Koichi Kobayashi1

Departments of 1Surgery and 2Medicine, Keio University School of Medicine, Tokyo; and 3Department of Pulmonary Medicine and Clinical Immunology, Dokkyo University School of Medicine, Tochigi, Japan

Submitted 19 May 2004 ; accepted in final form 1 July 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with 125I-labeled albumin. After intratracheal instillation of 125I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of 125I-albumin in plasma/total amount of instilled 125I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H2O2-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.

acute lung injury; acute respiratory distress syndrome; reexpansion pulmonary edema; Rho; Rho-kinase

REEXPANSION OF A COLLAPSED lung occasionally causes acute lung injury (ALI) known as reexpansion pulmonary edema (3, 17, 18). The main characteristic of this disorder is an increase in pulmonary microvascular permeability (24, 40). Vascular endothelial permeability is related to the cellular cytoskeleton. An increase in vascular permeability is accompanied by a reorganization of the actin-based cytoskeleton and contraction of endothelial cells, resulting in the formation of an intercellular gap (7, 28). A calmodulin-dependent myosin light chain kinase (MLCK) contributes to the regulation of cell contraction and endothelial cell permeability. Activation of MLCK causes the phosphorylation of the myosin light chains, resulting in the contraction of actomyosin, in the formation of stress fibers and in subsequent hyperpermeability of endothelial cells (8). A signaling pathway of the small GTPase Rho and its target protein, Rho-kinase (Rho-associated coiled-coil-forming protein kinase; ROCK), also regulates the contraction of endothelial cells (5). The activation of Rho leads to the phosphorylation of the myosin binding subunit of myosin light chain phosphatase via the action of ROCK, inactivating the myosin light chain phosphatase and blocking the dephosphorylation of myosin light chains (16). Continued phosphorylation of myosin light chains results in sustained cell contraction.

In studies in isolated rat lungs, a MLCK inhibitor attenuated the injury associated with ischemia-reperfusion (15) and that induced by ventilators (23). These observations suggest that the intracellular signal transduction that regulates the cellular cytoskeleton is involved in the pathophysiology of some forms of ALI. However, the role of the Rho/ROCK-mediated pathway in reexpansion pulmonary edema in vivo remains unclear, although more attention has been paid to the role of alveolar epithelial injury in the pathophysiology of ALI (38). The Rho/ROCK-mediated pathway has been implicated in the organization of perijunctional actin and in the regulation of permeability of the tight junction of the epithelial cells (6, 22). Therefore, we hypothesized that the Rho/ROCK-mediated pathway is involved in the changes in alveolar endothelial and epithelial barrier permeability in reexpansion pulmonary edema.

Y-27632, a highly selective inhibitor of ROCK in vitro and in vivo (36), was found useful to examine the role of the Rho/ROCK-mediated pathway in vivo (11, 29). To test our hypothesis, we examined the inhibitory effect of Y-27632 in our rabbit model of reexpansion lung injury (20). We measured the flux of transvascular albumin as an indicator of endothelial permeability (10). Alveolar epithelial permeability was assessed by measuring the bidirectional flux of albumin across the alveolar epithelial barrier (10, 30, 39). We also performed in vitro experiments in human pulmonary artery endothelial cell monolayers to study the effects of Y-27632 on hyperpermeability and endothelial cytoskeletal rearrangement induced by H2O2. These experiments were performed since reactive oxygen species appear to be involved in the injury to pulmonary endothelial cells observed in reexpansion pulmonary edema (12). We observed morphological changes in the microfilament cytoskeleton using a rhodamine-phalloidin stain (25). The main objective of this study was to ascertain the role of the Rho/ROCK pathway in the increased alveolar endothelial and epithelial barrier permeability present in reexpanded lungs.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The experiments were performed in 45 male Japanese White rabbits, weighing between 900 and 1,800 g. All procedures were reviewed and approved by the Laboratory Animal Care Panel of Keio University. The animals were divided into the following three groups: 1) a reexpansion group (RE), 2) a Rho-kinase inhibitor pretreatment group (RE + Y), which received 10 mg/kg Y-27632 intraperitoneally before reexpansion of the left lung, and 3) a sham-operated group (sham) that underwent thoracotomy only. The surgical techniques have been previously reported in detail (20). Briefly, general anesthesia was induced with intramuscular ketamine (75 mg/kg) and xylazine (5 mg/kg). A tracheostomy was performed, and a 3.5-mm-diameter Portex LTD tracheal cannula (Hythe, Kent, UK) was inserted. The rabbits were mechanically ventilated (SN-480–7; Shinano, Tokyo, Japan) with room air at 30 breaths/min and a tidal volume of 10 ml/kg.

In the RE and RE + Y groups, after completion of the left posterolateral thoracotomy, the left main bronchus was clamped with an atraumatic vascular clip (Vascu-statt; Scanlan International, St. Paul, MN) to produce complete collapse of the left lung by absorption of the alveolar gas in the pulmonary circulation. A 4–0 nylon string attached to the clip was exteriorized for later retraction. In the sham group, a left posterolateral thoracotomy was performed, and the left main bronchus was dissected from the surrounding tissue. The chest wall was closed. The rabbits were allowed to recover for 36 h before the experiments began. The tracheal cannula was removed after the recovery of spontaneous breathing.

Experimental Protocol

Experiment 1. Twenty-one animals were divided into three groups listed above to measure endothelial and epithelial permeability. The protocol for experiment 1 is provided in Fig. 1. Endothelial permeability was assessed from the transvascular flux of 125I-labeled albumin administered intravenously. Epithelial permeability was measured as the flux of 125I-albumin from intravascular space to airspace. To correct blood contamination in the lung tissue or bronchoalveolar lavage (BAL) fluid samples, 131I-albumin was injected intravenously. On the day of experiments, the rabbits were sedated with intramuscular ketamine (100 mg/kg) 30 min before the experiment and placed in the supine position, and a 24-gauge catheter was inserted in an ear vein. 125I-labeled BSA (2 ml of 37 kBq/ml; Life Science Products, Boston, MA) was injected intravenously to assess the pulmonary extravasated albumin in all experimental groups. In the RE and RE + Y groups, 10 min after 125I-albumin injection, the left main bronchus was reopened by retracting the string. At this time, a 9-Fr drainage chest tube (Sumitomo Bakelite, Tokyo, Japan) was inserted in the left pleural cavity, and continuous suction at –10 cmH2O was applied through a chest tube to reinflate the collapsed lung. In the RE + Y group, 10 mg/kg Y-27632 were administered in the peritoneal cavity 30 min before reopening the left main bronchus. An identical volume of saline was injected intraperitoneally in the sham and RE groups. Room air breathing resumed spontaneously. To prevent hypotension and dehydration, 50 ml/kg normal saline were infused intravenously throughout the period of continuous suction. 131I-labeled human serum albumin (2 ml of 37 kBq/ml; Daiichi Radioisotope Laboratories, Tokyo, Japan) was injected intravenously 115 min after reopening the left main bronchus. We used 131I-albumin as a plasma volume marker to correct for residual blood content in excised lung tissue samples. Heparin (2,000 IU iv) was injected simultaneously. The animals were killed 120 min after reopening the left main bronchus by injection of 100 mg pentobarbital intravenously. The chest was immediately opened, 5 ml blood were collected by cardiac puncture, each pulmonary hilus was clamped at the end of expiration, and the lungs were removed and divided into superior and inferior lobes. Blood and superior lobe lung samples were placed in a preweighed glass tube for measurements of weight and radioactivity.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 1. Protocol for experiment 1. The experimental groups consist of the following: sham-operated group (Sham), reexpansion group (RE), and Rho-kinase inhibitor pretreatment group (RE + Y). Sham group, only thoracotomy was performed. RE group, left main bronchus was declamped after 36 h of clamping, and the collapsed lung was reexpanded for 2 h. RE + Y group, 10 mg/kg specific Rho-kinase inhibitor (Y-27632) was administered in the peritoneal cavity 30 min before reopening the left main bronchus. 125I-albumin was injected iv 10 min before reopening the left main bronchus to assess transvascular permeability. 131I-albumin was injected iv 5 min before death to correct blood contamination.

 
Total blood cells were counted with a Sysmex K-1000 counter adapted for rabbit cells (Sysmex, Kobe, Japan). Differential counts of 200 cells were performed on glass slide smears stained by a modified Wright's stain (Diff-Quick; American Scientific, McGraw Park, IL).

BAL was performed with 10 ml normal saline through a catheter secured within the bronchus of the excised inferior lobe. The volume of fluid recovered from the left lung of the sham, RE, and RE + Y groups were 7.9 ± 0.3, 8.1 ± 0.4, and 8.0 ± 0.5 ml, respectively. The corresponding volumes recovered from the right lung were 7.9 ± 0.4, 8.3 ± 0.3, and 8.3 ± 0.5 ml, respectively. There was no significant difference in the recovery of BAL fluid among the groups. The fluid was centrifuged at 400 g and 4°C for 10 min. The supernatant was used for gamma counting of 125I and 131I to measure the pulmonary epithelial permeability, and for the analysis of neutrophil elastase activity and malondialdehyde (MDA) concentration. The cell pellet was resuspended in 1 ml saline, and the cells were counted by a modified hemocytometer method (Unopet Microcollection System; Becton-Dickson, Rutherford, NJ). Cell differential in BAL was counted in smears obtained by cytocentrifugation. Differential counts were made on 200 cells from smears stained with a modified Wright's stain.

In nine additional animals, divided into the three experimental groups and treated similarly, the mean arterial pressure was measured with a polygraph (Nihon Koden, Tokyo, Japan) via a catheter inserted in the carotid artery.

Experiment 2. Twelve animals were divided among the three experimental groups described earlier to measure the epithelial permeability. Epithelial permeability was measured as the flux from the airspace to intravascular space of 125I-albumin instilled in the left lung. On the day of experiments, 5 ml blood were collected from a catheter inserted in the right carotid artery, heparinized, and centrifuged to obtain plasma. The blood was replaced with an equal volume of Ringer lactate. After the onset of continuous suction (5 min), the rabbits were placed in the left lateral decubitus position to facilitate the collection of liquid in the left lung. Autologous plasma (2 ml/kg) containing 3 mg Evan's blue dye and 212 kBq 125I-labeled BSA were instilled in the left inferior lobe over 5 min with an 18-gauge tube advanced through the tracheostomy, as previously described (30, 39) We confirmed that most of the dye was distributed in the left lung after death of the animal. Blood samples were obtained for measurements of 125I-albumin activity at baseline and 10, 30, 60, 90, and 120 min after instillation. Because, clinically, the onset of reexpansion pulmonary edema takes place immediately or within a few hours (17), we chose these time points of observation to examine the early changes in flux of albumin from lung airspace to intravascular space.

Pulmonary Endothelial Permeability

Pulmonary endothelial permeability was calculated as the ratio of 125I-albumin per unit weight of lung tissue extravascular space over that in plasma (T/P ratio) in the left and right superior lobes. The activities of 125I and 131I in superior lobe parenchyma and blood were counted with a gamma counter (ARC-300; Aloca, Tokyo, Japan), with appropriate corrections for crossover between radionuclides. The extravascular 125I-albumin in the superior lobe was calculated by subtracting the amount of intravascular 125I-albumin from the total 125I-albumin in the lung tissue sample. The intravascular 125I-albumin in the superior lobe was corrected by calculating the ratio of 125I to 131I counts in the unit weight of blood and 131I-albumin in the unit weight of superior lobe parenchyma. We assumed that all of the 131I-albumin was confined to the circulation, since the effect of leakage of the plasma volume marker in the extravascular space is negligible when calculating T/P (13). Therefore, the 131I counts must reflect the residual blood in the lung tissue sample. The following formula was used to calculate the T/P ratio (10)

(1)
where 125I tissue is 125I cpm from a unit weight of superior lobe lung tissue, 125I blood is 125I cpm from a unit weight of blood, 131I tissue is 131I cpm from a unit weight of superior lobe lung tissue, 131I blood is 131I cpm from a unit weight of blood, and Hct is hematocrit.

Pulmonary Epithelial Permeability

Pulmonary epithelial permeability was ascertained by two separate methods used to measure the bidirectional flux of albumin across the alveolar epithelial barrier. 1) In experiment 1, the flux of albumin from intravascular space to lung airspace was assessed by measuring the BAL fluid-to-plasma 125I-albumin concentration ratio (B/P ratio). We hypothesized that, if epithelial permeability was increased, the amount of 125I-albumin entering the airspace from the circulation would increase, thus increasing the amount of 125I-albumin collected in BAL fluid. Blood contamination in BAL fluid was also corrected by 131I activity of BAL fluid (10). The following formula was used to calculate the B/P ratio (10)

(2)
where 125I BAL is 125I cpm from a unit weight of BAL fluid and 131I BAL is 131I cpm from a unit weight of BAL fluid.

2) In experiment 2, the flux of albumin from airspace to intravascular space, the plasma leak (PL) index, was calculated as the ratio of 125I-albumin in circulating plasma to that instilled in the left lung, a modification of a previously described method (30, 39). The amount of 125I-albumin in circulating plasma was calculated by assuming the plasma volume as volume (liter) = body weight (kg) x 0.07(1 – hematocrit) (30, 39).

Extravascular Lung Water

Extravascular lung water was measured as the blood-free wet-to-dry lung weight ratio (W/D). The superior lobe parenchyma and blood samples were weighed immediately after excision, dried for 72 h in a vacuum oven (DP22; Yamato, Tokyo, Japan) at 90°C and 200 mmHg, and then weighed again. We estimated the weight of residual blood contained in the tissue samples by counting 131I in the tissue and blood samples obtained at death. Because we assumed that all of the 131I-albumin was confined to the circulation, we were able to correct for the wet weight of the blood-free tissue sample. In addition, using the W/D ratio of the blood sample, we were also able to correct for the dry weight of the residual blood in the lung tissue sample. We calculated the dry weight of blood-free lung tissue by subtracting the weight of the residual blood from that of lung tissue sample. This allowed us to calculate the blood-free W/D ratio of each superior lobe lung tissue sample.

Neutrophil Elastase Activity and MDA Concentration in the BAL Fluid

Neutrophil elastase activity was determined with the highly specific synthetic substrate N-methoxysuccinli-Ala-Ala-Pro-Val p-nitroanilline by the method of Yoshimura et al. (42). MDA was measured with a BIOXYTECH LPO-586 kit (Oxis International, Portland, OR) by the method of Gerard-Monnier et al. (9). Briefly, N-methyl-2-phenylindole was added to the BAL fluid, followed by the addition of 12 N HCl, and incubated at 45°C for 60 min. After centrifugation at 2,500 g at 4°C for 10 min, the absorbance of the clear supernatant was measured at 586 nm. The standard curve was constructed with an MDA standard, and the MDA concentration was expressed in micromoles per liter.

Histopathological Examination

Histopathological examinations were performed to assess pulmonary neutrophil sequestration. Twelve animals were divided among the three experimental groups described earlier, and treated similarly. The right and left lungs were fixed by inflation with formalin, gravimetrically instilled at 25 cmH2O. The lung was fixed for ≥48 h before the preparation of 2- to 5-mm sagittal sections embedded in paraffin, from which 5-µm sections were sliced and stained with hematoxylin and eosin. The number of neutrophils was counted under oil at x1,000 magnification among 60 randomly selected fields per rabbit. The neutrophil count was divided by the number of alveoli in the corresponding field to compensate for variable lung inflation (34).

In Vitro Permeability Study

Human pulmonary artery endothelial cells in their fourth passage were obtained from KURABO Industries (Osaka, Japan) and were cultured in a humidified 5% CO2 atmosphere with RPMI-1640 medium supplemented with 100 mg/ml streptomycin, 100 U/ml penicillin, and 10% heat-inactivated, endotoxin free FBS (GIBCO-BRL Life Technologies, Grand Island, NY). Monolayers of endothelial cells were prepared on filters as previously described (41). In brief, 12-mm diameter Millicell-HA tissue culture plate well inserts were obtained from Millipore (Bedford, MA). The inserts consist of a surfactant-free 0.45-µm-pore size microporous cellulose membrane filter sealed to a cylindrical polystyrene holder. They were incubated for 3 h with 7 µg/cm2 human fibronectin (Sigma Chemical. St. Louis, MO) at 37°C to facilitate the cell attachment. The fibronectin solution was aspirated, and the human pulmonary artery endothelial cells suspended in the culture medium were seeded on the membrane filter at a density of 4 x 105 cells/insert (upper chamber). The inserts were placed in a six-well culture plate (Falcon; Becton-Dickinson, Lincoln Park, NJ), with each well filled with 2 ml culture medium, and incubated at 37°C in a humidified 5% CO2 atmosphere for 2 wk before the measurements of permeability.

To assess the permeability, we measured the albumin transferred across the monolayers of human pulmonary artery endothelial cells cultured on a porous filter. These monolayers were incubated at 37°C for 30 min in the culture medium containing the Y-27632 solution in concentrations of 1 and 10 µM, in a humidified 5% CO2 atmosphere. A 0.50-mM H2O2 solution was then added to the upper chamber. The culture medium was aspirated 1 h later, and 500 µl PBS containing 0.1% BSA were added to the upper chamber. The insert was placed in 1 well of a 24-well culture plate (Falcon; Becton-Dickinson), where each well was filled with 0.7 ml PBS alone. After incubation for 20 min, the insert was removed from the well, and the albumin concentration of the lower chamber was measured with a Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA).

Morphological Observations Using Rhodamine-Phalloidin Stain

The changes in the actin microfilament cytoskeleton of the monolayers of human pulmonary artery endothelial cells grown on filters were examined with a rhodamine-phalloidin stain (Molecular Probes, Eugene, OR), as described previously (25), and photographed with a fluorescence microscope equipped with epi-illumination (Nikon Optiphot; Nikon, Garden City, NY).

Statistical Analysis

All data are expressed as means ± SE. One-way ANOVA followed by Fisher's least significant difference test was used for among-groups and between-lung comparisons. One-way ANOVA with repeated-measurements analysis was used to estimate the significance of PL and mean arterial pressures. Differences with P values <0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In Vivo Study

The measurements of 125I and 131I activities in blood, superior lobe lung tissue, and BAL fluid are shown in Table 1. The T/P ratios are shown in Fig. 2. In the left or reexpanded lung, pretreatment with Y-27632 inhibited the increase in T/P ratio after reexpansion. Endothelial permeability in the RE group was significantly higher than in the sham and RE + Y groups. In contrast, in the right lung, there was no significant difference in the T/P ratio among the three groups.


View this table:
[in this window]
[in a new window]
  		Table 1. 125I and 131I activities in blood, lung, and BAL fluid

 


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 2. The tissue-to-plasma (T/P) ratio, as an index of pulmonary endothelial permeability. The left lung is the test lung, and the right lung is the control. Values are means + SE; n = 7 rabbits in each group. P < 0.01 vs. sham group (*) and vs. RE + Y group (#).

 
Figure 3 shows the B/P ratio measured in each lung. In the left lung of the RE group, the albumin flux in the airspace was significantly higher than in the left lung of the sham and the RE + Y groups. There was no difference among groups in the B/P ratio in the right lung.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. The bronchoalveolar lavage fluid (BALF)-to-plasma (B/P) ratio, as an index of pulmonary epithelial permeability. The left lung is the test lung, and the right lung is the control. Values are means + SE; n = 7 in each group. P < 0.05 vs. sham group (*) and vs. RE + Y group (#).

 
The PL from airspace to intravascular space up to 120 min after instillation of 125I-albumin in the left lung is shown in Fig. 4. PL was significantly higher at 10 min after instillation of 125I-albumin in the RE group than in the other two groups. Furthermore, in the RE group, the W/D ratio was significantly greater than in the RE + Y group (Fig. 5).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 4. Changes in plasma leak (PL) index up to 2 h. Values are means ± SE; n = 4 in each group. P < 0.01 vs. sham group (*) and vs. RE + Y groups (+). #P < 0.05 vs. RE + Y group.

 


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 5. Lung wet-to-dry (W/D) ratio, an index of pulmonary edema. The left lung is the test lung, and the right lung is the control. Values are means + SE; n = 7 in each group. P < 0.05 vs. the sham group (*) and vs. the RE + Y group (#).

 
The mean total peripheral neutrophil counts at the end of experiments are shown in Table 2. There were no significant differences among the groups. On light microscopy, the number of neutrophils per alveolus in the left lung of both the RE and RE + Y groups was significantly greater than in the sham group (Fig. 6). There was no difference, in the right lung, in neutrophil counts among the groups. The neutrophil count in the left lung BAL fluid in the RE and RE + Y groups was more than twofold greater than in the sham group, although the difference did not reach statistical significance (Fig. 7).


View this table:
[in this window]
[in a new window]
  		Table 2. Total and differential white blood cell counts at time of animal death

 


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 6. Tissue neutrophil counts per alveolus in lung. Values are means ± SE; n = 4 in each group. *P < 0.01 vs. sham group.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 7. Neutrophil counts (x105/ml) in bronchoalveolar lavage (BAL) fluid. Values are means ± SE; n = 7 in each group.

 
The BAL fluid neutrophil elastase activity in the left lung of the RE and RE + Y groups was significantly higher than in the left lung of the sham group, whereas there was no significant difference in neutrophil elastase activity among the three groups in the right lung (Table 3). In the left lung, the MDA concentration of BAL fluid was significantly higher in the RE than in the sham group, although it was similar in the RE and the RE + Y groups (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Malondialdehyde concentration and neutrophil elastase activity in BAL fluid

 
Mean arterial pressure did not differ significantly among the experimental groups (Table 4).


View this table:
[in this window]
[in a new window]
  		Table 4. Mean arterial pressures

 
Permeability in Pulmonary Endothelial Cell Monolayers

Figure 8 shows the endothelial permeability index estimated from the albumin transfer across human pulmonary endothelial cell monolayers. H2O2 increased the permeability of the monolayer above control levels, and treatment with Y-27632 in concentrations of 1 and 10 µM significantly attenuated the increase induced by H2O2.



View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. Effect of Y-27632 on the H2O2-induced (0.50 mM) increase in permeability in pulmonary endothelial cell monolayers. Values are means ± SE; n = 5 in each group. *P < 0.005 vs. H2O2 + Y-27632 (0 µM). #P < 0.0001 vs. H2O2 + Y-27632 (0 µM).

 
Morphological Changes

We used four monolayers in each group and show representative findings in Fig. 9. Close cell-to-cell contact was present in the control preparations of human pulmonary artery endothelial cells (Fig. 9A). Treatment with H2O2 caused the development of randomly oriented stress fibers, cell contraction, and intercellular gaps (Fig. 9B), and treatment with Y-27632 mitigated these changes (Fig. 9C).



View larger version (77K):
[in this window]
[in a new window]
  		Fig. 9. Morphological changes in pulmonary endothelial cells. Rhodamine-phalloidin stain of monolayers. A: control; B: monolayers 60 min after treatment with 0.50 mM H2O2; C: pretreatment with Y-27632 (10 µM) for 30 min before H2O2 treatment. Scale bar = 20 µm.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We examined the role of the Rho/ROCK-mediated pathway in the changes in vascular endothelial and alveolar epithelial permeability, in a new rabbit model of reexpansion pulmonary edema. Treatment with Y-27632, a ROCK-specific inhibitor, attenuated ALI after reexpansion of a lung collapsed for 36 h. We used the 125I-albumin T/P ratio as an index of vascular endothelial permeability, measured the bidirectional flux of albumin across the alveolar epithelium by a modification of the method described by Smedira et al. (30) and Wiener-Kronish et al. (39), and calculated the 125I-albumin B/P ratio and the PL index as indexes of epithelial permeability. These indexes were all increased in the reexpanded lung, increases that were suppressed by pretreatment with Y-27632 (Figs. 2–4). However, Y-27632 neither prevented the accumulation of neutrophils nor inhibited the effects of neutrophil elastase activity and MDA production in the reexpanded lung (Figs. 6 and 7 and Table 2).

The T/P ratio represents the amount of 125I-albumin that traverses the pulmonary vascular endothelium per unit of time. The transendothelial flux of albumin is mainly determined by endothelial permeability, capillary surface area, and capillary pressure (4, 35). Although the administration of Y-27632 did not lower the systemic blood pressure in this study, it may have lowered the pulmonary artery and capillary pressures and increased the capillary surface area, since it relaxes smooth muscles by its ROCK inhibitory action (2). However, if the intraperitoneal administration of Y-27632 had changed the T/P ratio by relaxing the smooth muscle in this model, the ratio in the right lung should have differed among the groups. Therefore, the suppression of the increased T/P by Y-27632 pretreatment in the reexpanded lung was mainly the result of suppression of an increased pulmonary vascular permeability to albumin. These findings confirm that the Rho/ROCK pathway may contribute to the increase in vascular endothelial permeability that develops in reexpanded lungs.

In patients with reexpansion pulmonary edema, the concentrations of neutrophil chemoattractant, such as IL-8 and leukotriene B4, are increased in edema fluid and are accompanied by higher numbers of pulmonary neutrophils (19, 31). In addition, the administration of antioxidants before lung reexpansion mitigates the development of edema, supporting the hypothesis that reactive oxygen species produced by pulmonary neutrophils may play important roles in the development of reexpansion pulmonary edema (12). In our study, neutrophils accumulated together with a rise in BAL fluid neutrophil elastase activity and MDA concentration, which reflects lipid peroxidation caused by reactive oxygen species in the reexpanded lung (Figs. 6 and 7 and Table 2). These observations suggest that fluid mediators released from activated neutrophils, such as elastase and/or reactive oxygen species, contribute to the development of lung reexpansion injury.

Several studies, including ours, suggest that reexpansion pulmonary edema is associated with an increase in pulmonary vascular endothelial permeability (20, 24, 40). However, few have examined the alveolar epithelial permeability in this disorder. Pulmonary edema often develops rapidly after reexpansion, and the protein concentration in edema fluid is increased (17, 19, 31), suggesting a concomitant increase in epithelial permeability. Injury to the alveolar epithelial barrier markedly influences the severity of ALI (38). By modifying the method of Smedira et al. (30) and Wiener-Kronish et al. (39), we were able to examine the epithelial permeability in reexpanded lungs by measuring the bidirectional flux of 125I-albumin through the alveolar septal barrier. We found that both the B/P and PL index were increased, which is consistent with an increase in epithelial permeability in the reexpanded lung. The simultaneous increase in vascular endothelial and alveolar epithelial permeability is likely to be the source of severe and acute disease manifestations at the very onset of reexpansion pulmonary edema. Because the PL index began to rise immediately after reexpansion (Fig. 4), the increase in epithelial permeability may occur immediately after reexpansion. It has been reported that alveolar epithelium is more resistant to injury than the pulmonary vascular endothelium (26). The immediate increase in PL index after reexpansion may be an indication that the mechanical stretch of the alveolar epithelial barrier plays a role in the increased epithelial permeability of the reexpanded lung (Fig. 4).

Y-27632 interacts with ROCK in human neutrophils to suppress the production of superoxide and the chemotactic peptide-induced cell motile function in vitro (14, 21). In the rat liver, Y-27632 attenuated ischemia and reperfusion injury by limiting the infiltration by neutrophils and rise in tissue MDA concentration (32). However, in the present study, pretreatment with Y-27632 did not prevent the accumulation of neutrophils and production of reactive oxygen species in the lung reexpanded after 36 h of collapse. In a preliminary study, we observed neutrophil influx in collapsed lungs before their reexpansion. We also have reported that the production of IL-8 by macrophages and alveolar epithelial cells, which plays an important role in the migration of neutrophils in reexpanded lungs, starts at the time of lung collapse (20). Treatment instituted immediately before reexpansion may be insufficient to inhibit recruitment and activation of neutrophils.

The increase in microvascular permeability is associated with the formation of an endothelial paracellular gap. An actin-myosin contraction of the endothelial cells is important to the regulation of paracellular gap formation (8, 28). It has been reported that the small GTPase Rho plays an important role in the regulation of actomyosin contractile elements (27, 33). Rho and its downstream effector, ROCK, are involved in signal transduction in endothelial cells, linking extracellular stimuli to a dynamic rearrangement of cytoskeletal actin (33). The activation of Rho, via the action of ROCK, causes the phosphorylation of the myosin-binding subunit of myosin light chain phosphatase, inactivating the latter and increasing the phosphorylation of myosin light chains, actomyosin contraction, and endothelial cell barrier dysfunction (1, 5, 16). In a cultured cell model of thrombin-induced endothelial cell barrier dysfunction, the ROCK inhibitor Y-27632 decreases endothelial hyperpermeability and suppresses the reorganization of F-actin and the formation of intercellular gaps (37). Chiba et al. (2) also reported a suppressive effect of Y-27632 on H2O2-induced pulmonary edema in isolated rabbit lungs. In cultured pulmonary endothelial cells, MLCK contributes to H2O2-induced actin rearrangement (43). These observations suggest that the Rho/ROCK pathway is involved in the increased vascular endothelial permeability induced by reactive oxygen species. In this study, Y-27632 attenuated significantly both the increase in endothelial permeability and the reorganization of F-actin. These observations suggest that Y-27632 downregulates the Rho/ROCK signal transduction pathway in endothelial cells and suppresses the hyperpermeability induced by reactive oxygen species in the reexpanded lung by inhibiting the reorganization of F-actin, subsequently blocking the contraction of vascular endothelial cells. In addition, Rho protein regulates the epithelial tight junction and perijunctional actin organization (6, 22). Y-27632 may influence epithelial cells by protecting the cellular barrier, although this was not examined in the present study.

In summary, this is the first report of the involvement of the Rho/ROCK-mediated pathway in the increase in pulmonary vascular endothelial and alveolar epithelial permeability in reexpansion pulmonary edema. Y-27632 may be effective in treating reexpansion pulmonary edema and other types of ALI associated with an increase in alveolar barrier permeability.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported, in part, by Grant-in-Aid 11770744 for Fundamental Scientific Research from the Ministry of Education, Science, and Culture of Japan (to M. Sawafuji).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Sawafuji, Dept. of Surgery, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (e-mail: msawafuji{at}nifty.com)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amano M, Chihara K, Kimura K, FukataY, Nakamura N, Matsuura, and Kaibuchi K. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 275: 1308–1311, 1997.[Abstract/Free Full Text]
  2. Chiba Y, Ishii Y, Kitamura S, and Sugiyama Y. Activation of Rho is involved in the mechanism of hydrogen-peroxide-induced lung edema is isolated perfused rabbit lung. Microvasc Res 62: 164–171, 2001.[CrossRef][ISI][Medline]
  3. Childress ME, Moy G, and Mottram M. Unilateral pulmonary edema resulting from treatment of spontaneous pneumothorax. Am Rev Respir Dis 104: 119–121, 1971.[Medline]
  4. Dull RO, Jo H, Sill H, Hollis TM, and Tarbell JM. The effect of varying albumin concentration and hydrostatic pressure on hydraulic conductivity and albumin permeability of cultured endothelial monolayers. Microvasc Res 41: 390–407, 1991.[CrossRef][ISI][Medline]
  5. Essler M, Amano M, Kruse HJ, Kaibuchi K, Weber PC, and Aepfelbacher M. Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells. J Biol Chem 273: 21867–21874, 1998.[Abstract/Free Full Text]
  6. Fujita H, Katoh H, Hasegawa H, Yasui H, Aoki J, Yamaguchi Y, and Negishi M. Molecular decipherment of Rho effector pathways regulating thght-junction permeability. Biochem J 346: 617–622, 2000.
  7. Garcia JG, Siflinger-Birnboim A, Bizios R, Del Vecchio PJ, Fenton 2nd JW, and Malik AB. Thrombin-induced increases in albumin permeability across the endothelium. J Cell Physiol 128: 96–106, 1986.[CrossRef][ISI][Medline]
  8. Garcia JG, Davis HW, and Patterson CE. Regulation of endothelial cell gap formation and barrier dysfunction: Role of myosin light chain phosphorylation. J Cell Physiol 163: 510–522, 1995.[CrossRef][ISI][Medline]
  9. Gerard-Monnier D, Erdelmeier I, Regnard K, Moze-Henry N, Yaden JC, and Chaudiere J. Reactions of 1-methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation. Chem Res Toxicol 11: 1176–1183, 1998.[CrossRef][ISI][Medline]
  10. Ishizaka A, Hasegawa N, Sakamaki F, Tasaka S, Nakamura H, Kishikawa K, Yamada A, Obata T, Sayama K, Urano T, and Kanazawa M. Effects of ONO-1078, a peptide leukotriene antagonist, on endotoxin-induced acute lung injury. Am J Respir Crit Care Med 150: 1325–1331, 1994.[Abstract]
  11. Itoh K, Yoshioka K, Akedo H, Uehata M, Ishizaki T, and Narumiya S. An essential part for Rho-associated kinase in the transcelular invasion of tumor cells. Nat Med 5: 221–225, 1999.[CrossRef][ISI][Medline]
  12. Jackson RM, Veal CF, Alexander CB, Brannen AL, and Fulmer JD. Re-expansion pulmonary edema. A potential role for free radicals in its pathogenesis. Am Rev Respir Dis 137: 1165–1171, 1988.[Medline]
  13. Kanazawa M, Hasegawa N, Urano T, Sayama K, Tasaka S, Sakamaki F, Nakamura H, Waki Y, Terashima T, Fujishima S, and Ishizaka A. Regional lung hematocrit variation and assessment of acute lung injury. J Appl Physiol 77: 567–573, 1994.[Abstract/Free Full Text]
  14. Kawaguchi A, Ohmori M, Harada K, Tsuruoka S, Sugimoto K, and Fujimura A. The effect of a Rho kinase inhibitor Y-27632 on superoxide production, aggregation and adhesion in human polymorphonuclear leukocytes. Eur J Pharmacol 8: 203–208, 2000.
  15. Khimenko PL, Moore TM, Wilson PS, and Taylor AE. Role of calmodulin and myosin light-chain kinase in lung ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol 271: L121–L125, 1996.[Abstract/Free Full Text]
  16. Kimura K, Ito M, Amano M, Chihara K, Fukata Y, Nakafuku M, Yamamori B, Feng J, Nakano T, Ogawa K, Iwamatsu A, and Kaibuchi K. Regulation of myosin phosphatase by Rho and Rho-assosiated kinase (Rho-kinase). Science 273: 245–248, 1996.[Abstract]
  17. Mahfood S, Hix WR, Aaron BL, Blaes P, and Watson DC. Reexpansion pulmonary edema. Ann Thorac Surg 45: 340–345, 1988.[Abstract]
  18. Matsuura Y, Nomimura T, Murakami H, Matsushima T, Kakehashi M, and Kajihara H. Clinical analysis of reexpansion pulmonary edema. Chest 100: 1562–1566, 1991.[Abstract/Free Full Text]
  19. Nakamura H, Ishizaka A, Sawafuji M, Urano T, Fujishima S, Sakamaki F, Sayama K, Kawamura M, Kato R, Kikuchi K, Kanazawa M, Kobayashi K, and Kawashiro T. Elevated levels of interleukin-8 and leukotriene B4 in pulmonary edema fluid of a patient with reexpansion pulmonary edema. Am J Respir Crit Care Med 149: 1037–1040, 1994.[Abstract]
  20. Nakamura M, Fujishima S, Sawafuji M, Ishizaka A, Oguma T, Soejima K, Matsubara H, Tasaka S, Kikuchi K, Kobayashi K, Ikeda E, Sadick M, Hebert CA, Aikawa N, Kanazawa M, and Yamaguchi K. Importance of interleukin-8 in the development of reexpansion lung injury in rabbits. Am J Respir Crit Care Med 161: 1030–1036, 2000.[Abstract/Free Full Text]
  21. Niggli V. Rho-kinase in human neutrophils: a role in signaling for myosin light chain phosphorylation and cell migration. FEBS Lett 445: 69–72, 1999.[CrossRef][ISI][Medline]
  22. Nusrat A, Giry M, Turner JR, Colgan SP, Parkos CA, Carnes D, Lemichez E, Boquet P, and Madara JL. Rho protein regulates tight junctions and perijunctional actin organization in polarized epithelia. Proc Natl Acad Sci USA 92: 10629–10633, 1995.[Abstract/Free Full Text]
  23. Parker JC. Inhibitors of myosin light chain kinase and phosphodiesterase reduce ventilator-induced lung injury. J Appl Physiol 89: 2241–2248, 2000.[Abstract/Free Full Text]
  24. Pavlin DJ, Nessly ML, and Cheney FW. Increased pulmonary vascular permeability as a cause of reexpansion edema in rabbits. Am Rev Respir Dis 124: 422–427, 1981.[ISI][Medline]
  25. Phillips PG and Tsan MF. Direct staining and visualization of endothelial monolayers cultured on synthetic polycarbonate filters. J Histochem Cytochem 36: 551–554, 1988.[Abstract]
  26. Pittet JF, Matthay MA, Pier G, Grady M, and Wiener-Kronish JP. Pseudomonas aeruginosa-induced lung and pleural injury in sheep. Differential protective effect of circulating versus alveolar immunoglobulin G antibody. J Clin Invest 92: 1221–1228, 1993.[ISI][Medline]
  27. Ridley AJ and Hall A. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399, 1992.[CrossRef][ISI][Medline]
  28. Shasby DM, Shasby SS, Sullivian JM, and Peach MJ. Role of endothelial cell cytoskelton in control of endothelial permeability. Circ Res 51: 657–661, 1982.[Abstract]
  29. Shimizu Y, Dobashi K, Iizuka K, Horie T, Suzuki K, Tukagoshi H, Nakazawa T, Nakazato Y, and Mori M. Contribution of small GTPase Rho and its target protein ROCK in a murine model of lung fibrosis. Am J Respir Crit Care Med 163: 210–217, 2001.[Abstract/Free Full Text]
  30. Smedira N, Gates L, Hastings R, Jayr C, Sakuma T, Pittet JF, and Matthay MA. Alveolar and lung liquid clearance in anesthetized rabbits. J Appl Physiol 70: 1827–1835, 1991.[Abstract/Free Full Text]
  31. Suzuki S, Tanita T, Koike K, and Fujimura S. Evidence of acute inflammatory response in reexpansion pulmonary edema. Chest 101: 275–276, 1992.[Abstract/Free Full Text]
  32. Takeda K, Jin MB, Fujita M, Fukai M, Sakurai T, Nakayama M, Taniguchi M, Suzuki T, Shimamura T, Furukawa H, and Todo S. A novel inhibitor of Rho-associated protein kinase, Y-27632, ameliorates hepatic ischemia and reperfusion injury in rats. Surgery 133: 197–206, 2003.[CrossRef][ISI][Medline]
  33. Tapon N and Hall A. Rho, Rac, and Cdc42 GTPases regulate the organization of the actin cytoskeleton. Curr Opin Cell Biol 9: 86–92, 1997.[CrossRef][ISI][Medline]
  34. Tasaka S, Ishizaka A, Urano T, Sayama K, Sakamaki F, Nakamura H, Terashima T, Waki Y, Soejima K, Oyamada Y, Fujishima S, and Kanazawa M. BCG-priming enhances endotoxin-induced acute lung injury independent of neutrophils. Am J Respir Crit Care Med 152: 1041–1049, 1995.[Abstract]
  35. Taylor AE and Drake RE. Fluid and protein movement across the pulmonary microcirculation. In: Lung Water and Solute Exchange, edited by Staub NC. New York, NY: Dekker, 1978 p. 129–182.
  36. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertention. Nature 389: 990–994, 1997.[CrossRef][Medline]
  37. van Nieuw Amerongen GP, van Delft S, Vermeer MA, Collard JG, and van Hinsbergh VW. Activation of RhoA by thrombin in endothelial hyper permeability: role of Rho kinase and protein tyrosine kinases. Circ Res 87: 335–340, 2000.[Abstract/Free Full Text]
  38. Ware LB and Matthay MA. The acute respiratory distress syndrome. N Engl J Med 342: 1334–1349, 2000.[Free Full Text]
  39. Wiener-Kronish JP, Sakuma T, Kudoh I, Pittet JF, Frank D, Dobbs L, Vasil ML, and Matthay MA. Alveolar epithelial injury and pleural empyema in acute P aeruginosa pneumonia in anethetized rabbits. J Appl Physiol 75: 1661–1669, 1993.[Abstract/Free Full Text]
  40. Wilkinson PD, Keegan J, Davies SW, Bailey J, and Rudd RM. Changes in pulmonary microvascular permeability accompanying re-expansion oedema: evedence from dual isotope scintigraphy. Thorax 45: 456–459, 1990.[Abstract]
  41. Yonemaru M, Kasuga I, Kusumoto H, Kiyokawa H, Kuwabara S, Ichinose Y, and Toyama K. Protein kinase inhibitor attenuates an increasa in endothelial monolayer permeability induced by tumor necrosis facter-alpha. Respirology 2: 63–69, 1997.[Medline]
  42. Yoshimura K, Nakagawa S, Koyama S, Kobayashi T, and Homma T. Roles of neutrophil elastase and superoxide anion in leukotriene B4-induced lung injury in rabbit. J Appl Physiol 76: 91–96, 1994.[Abstract/Free Full Text]
  43. Zhao Y and Davis HW. Hydrogen peroxide-induced cytoskeletal rearrangement in cultured pulmonary endothelial cells. J Cell Physiol 174: 370–379, 1998.[CrossRef][ISI][Medline]



This article has been cited by other articles:


Home page
 Circ. Res.Home page
G. P. van Nieuw Amerongen and V. W.M. van Hinsbergh
Endogenous RhoA Inhibitor Protects Endothelial Barrier
Circ. Res., July 6, 2007; 101(1): 7 - 9.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
289/6/L946    most recent
00188.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sawafuji, M.
Right arrow Articles by Kobayashi, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sawafuji, M.
Right arrow Articles by Kobayashi, K.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.