AJP - Lung Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 290: L1078-L1086, 2006. First published January 6, 2006; doi:10.1152/ajplung.00239.2005
1040-0605/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/L1078    most recent
00239.2005v1
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 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 Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takenaka, K.
Right arrow Articles by Yokoyama, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takenaka, K.
Right arrow Articles by Yokoyama, M.

Ventilator-induced lung injury is reduced in transgenic mice that overexpress endothelial nitric oxide synthase

Kaori Takenaka, Yoshihiro Nishimura, Teruaki Nishiuma, Akihiro Sakashita, Tomoya Yamashita, Kazuyuki Kobayashi, Miyako Satouchi, Tatsuro Ishida, Seinosuke Kawashima, and Mitsuhiro Yokoyama

Division of Cardiovascular and Respiratory Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Chuo-ku, Kobe, Japan

Submitted 1 June 2005 ; accepted in final form 28 December 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although mechanical ventilation (MV) is an important supportive strategy for patients with acute respiratory distress syndrome, MV itself can cause a type of acute lung damage termed ventilator-induced lung injury (VILI). Because nitric oxide (NO) has been reported to play roles in the pathogenesis of acute lung injury, the present study explores the effects on VILI of NO derived from chronically overexpressed endothelial nitric oxide synthase (eNOS). Anesthetized eNOS-transgenic (Tg) and wild-type (WT) C57BL/6 mice were ventilated at high or low tidal volume (VT; 20 or 7 ml/kg, respectively) for 4 h. After MV, lung damage, including neutrophil infiltration, water leakage, and cytokine concentration in bronchoalveolar lavage fluid (BALF) and plasma, was evaluated. Some mice were given N{omega}-nitro-L-arginine methyl ester (L-NAME), a potent NOS inhibitor, via drinking water (1 mg/ml) for 1 wk before MV. Histological analysis revealed that high VT ventilation caused severe VILI, whereas low VT ventilation caused minimal VILI. Under high VT conditions, neutrophil infiltration and lung water content were significantly attenuated in eNOS-Tg mice compared with WT animals. The concentrations of macrophage inflammatory protein-2 in BALF and plasma, as well as plasma tumor necrosis factor-{alpha} and monocyte chemoattractant protein-1, also were decreased in eNOS-Tg mice. L-NAME abrogated the beneficial effect of eNOS overexpression. In conclusion, chronic eNOS overexpression may protect the lung from VILI by inhibiting the production of inflammatory chemokines and cytokines that are associated with neutrophil infiltration into the air space.

nitric oxide; acute lung injury; mechanical ventilation; macrophage inflammatory protein-2; macrophage chemoattractant protein-1

VENTILATOR-INDUCED LUNG INJURY (VILI), which is one type of acute lung injury (ALI), has the characteristics of diffuse alveolar damage, namely, an initial vascular leak with a neutrophil-predominant inflammatory response (26, 32, 33). Positive-pressure mechanical ventilation (MV) can save the lives of patients with acute respiratory distress syndrome (ARDS), and recent approaches indicate that lung protective ventilator strategies can reduce mortality during ARDS (1). A large, multicenter randomized trial has shown that MV with a lower tidal volume results in lower mortality than conventional ventilation (1). On the other hand, large tidal volumes in animal models of VILI promote cytokine release (8, 34) and cause noncardiogenic pulmonary edema with accumulation of neutrophils (3, 5).

Among the inflammatory reactions that lead to injury, nitric oxide (NO) is an important factor that regulates microvascular permeability during the pathogenesis of ALI (6, 18). NO is synthesized from L-arginine by various isoforms of NO synthase (NOS) such as constitutive neuronal NOS (nNOS, or NOS1), inducible NOS (iNOS, or NOS2), and endothelial NOS (eNOS, or NOS3). Among the three isoforms, iNOS is believed to be the only isoform that is induced by systemic or local inflammation. eNOS is usually constitutively expressed as an intracellular protein in airway epithelial and pulmonary vascular endothelial cells (4, 14). Under physiological conditions, basal NO release from endothelial cells plays important roles in the inhibition of leukocyte attachment, maintenance of mast cell stability, and reduction of platelet aggregation (18) and also protects against microvascular permeability in intestinal vessels (16). Although the pathogenesis of ALI has been explored in many animal models, the role of NO in ALI remains controversial because it has pro- as well as anti-inflammatory effects (4). We reported that chronic eNOS overexpression in the mouse endothelium results in resistance to lipopolysaccharide (LPS)-induced lung injury (35). However, because of the overwhelming amount of iNOS-related NO produced in this model, the contribution of NO from eNOS transgenic expression was difficult to interpret. The present study explores the role of overloaded eNOS in a mouse model of VILI without treatment such as LPS. We established an MV model using eNOS transgenic mice without stimulation of iNOS expression and activity. To test the hypothesis that overloaded NO suppresses VILI through neutrophil chemoattractive protein, we assessed the release of cytokines and chemokines from eNOS-overexpressing mice and performed histological evaluations.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal preparation. We used 7- to 10-wk-old eNOS transgenic mice (Tg) and their wild-type (WT) littermate controls derived from the same genetic background (C57BL/6). The transgenic mice overexpress the bovine eNOS gene in the endothelium under the control of the preproendothelin-1 promoter (22). All animal experiments proceeded according to the Guidelines for Animal Experimentation at Kobe University Graduate School of Medicine.

Murine model of VILI. The mouse model of VILI was generated as described with modifications (2). Briefly, mice were anesthetized with pentobarbital sodium (50 mg/kg ip; Abbott Laboratories, Abbott Park, IL), and the trachea was exposed under sterile conditions. A 22-gauge ventilation cannula was inserted into the trachea and sutured. The mice were placed in the supine position on a warming device and then connected to a ventilator.

We selected two types of ventilators to establish an accurate tidal volume (VT). We used an HSE-Harvard MiniVent for the low VT study and a Harvard Apparatus (South Natick, MA) ventilator model 683 for the high VT study without intravascular cannulation. Blood pressure was measured using the tail-cuff method (model MK-1100; Muromachi Kikai, Tokyo, Japan) every 30 min during ventilation. The respiratory rate was adjusted to maintain pH between 7.30 and 7.45, according to arterial blood analysis. The control mice underwent tracheostomy without ventilation.

We initially examined the effect of various tidal volumes and respiratory periods. Because neutrophil recruitment into bronchoalveolar lavage fluid (BALF) was detected 4 h after ventilation, we selected this ventilation period for subsequent studies. We compared ventilation with low VT (7 ml/kg; respiratory rate of 100 breaths/min) and high VT (20 ml/kg; respiratory rate of 80 breaths/min). Whereas low VT resulted in a noninvasive ventilation tidal volume, as shown elsewhere (1), high VT used the same tidal volume that caused VILI in previous studies (6, 27). Both were maintained for 4 h at an inspiratory oxygen fraction of 0.21.

High VT resulted in a hyperventilation state [pH: 7.414 ± 0.035; arterial PO2 (PaO2): 117.1 ± 4.4 Torr; arterial PCO2 (PaCO2): 27.0 ± 1.6 Torr; n = 6] compared with low VT (pH: 7.314 ± 0.004; PaO2: 86.8 ± 11.4 Torr; PaCO2: 37.2 ± 5.8 Torr; n = 6) at the end of ventilation. Arterial blood gas data did not significantly differ between mouse groups.

The mice received 0.1 ml of 0.9% normal saline (intraperitoneally) immediately before MV started, followed by 0.1 ml of normal saline every hour thereafter. During ventilation, the mice were monitored every 30 min for adequate sedation, and pentobarbital sodium was administered as necessary. The tidal volume of the mice also was calculated by integrating airway flow during inspiration with a murine respiratory monitor (WinPULMOS II; M.I.P.S., Osaka, Japan). Some WT mice were given LPS (20 mg/kg ip; Sigma Chemical, St. Louis, MO) without MV as a positive control for iNOS induction.

To study the effects of chronic NOS inhibition on lung injury, some mice received N{omega}-nitro-L-arginine methyl ester (L-NAME; Sigma Chemical), a potent inhibitor of NOS, in drinking water ad libitum (1 mg/ml) for 1 wk before the experiment (24).

Histopathology. Four hours after MV, the mice were killed and the lungs and trachea were removed. The right main bronchus was tightly sutured, and the whole right lung was cut to determine the wet-to-dry weight ratio (W/D ratio). The left lung was infused at a pressure of 20 cmH2O with 10% buffered formalin, embedded in paraffin, sectioned at 5-µm thickness, and stained with hematoxylin and eosin. The histopathology of five random tissue sections from five or more lungs from each group was examined. A modified VILI histological scoring system was applied as described previously (2). Briefly, the following pathological processes were scored on a scale of 0 to 4: 1) alveolar congestion, 2) hemorrhage, 3) leukocyte infiltration or aggregation of neutrophils in the air space or vessel wall, and 4) thickness of the alveolar wall. A score of 0 represented normal lungs, and scores of 1, 2, 3, and 4 represented mild (<25%), moderate (25–50%), severe (50–75%), and very severe (>75%) lung involvement, respectively. The overall score was based on the sum of all scores. Two pathologists who were blinded to the treatment groups reviewed the degree of injury in three random sections of at least five lungs from each group of mice.

Lung water content. Lung water content related to lung injury was measured using the lung W/D ratio as described previously (27). Lung wet weight was determined immediately after removal, and the dry weight was determined after the lung was placed in an oven at 80°C for 48 h. The W/D ratio was calculated as the ratio of the wet weight to the dry weight (n = 6).

Bronchoalveolar lavage. After MV, the mice lungs were immediately perfused, and then BALF was obtained twice with 0.75 ml of saline (n = 6–8). Total cells in each sample were counted in 40-µl aliquots, using standard hematological procedures. The remaining fluid was centrifuged at 2,000 rpm for 5 min at 4°C, and cytokines were measured in the supernatants. The cell pellets were resuspended in saline, and slides were prepared by centrifugation at 400 rpm for 3 min in a Cytospin 2 (Shandon, Pittsburgh, PA). Specimens of BALF were stained.

ELISA for analysis of cytokines. Levels of tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-6, macrophage chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-2 were determined in plasma and in BALF supernatants by using ELISA kits. The TNF-{alpha}, IL-6, and MCP-1 kits were from Biosource International (Camarillo, CA), and the MIP-2 kit was from TECHNE (Minneapolis, MN). The sensitivity of these kits for TNF-{alpha}, IL-6, MCP-1, and MIP-2 were 3, 3, 9, and 1.5 pg/ml, respectively, and the values were mouse specific. The absorbance of each sample was measured at 450 nm with the use of a Multiskan JX microplate reader (Thermo Labsystems, Thermo Bio-analysis, Tokyo, Japan).

Analysis of eNOS and iNOS expression. To determine the amount of NOS protein in the lung after injury, we analyzed the left lungs after lavage as previously described (22). Lung protein (50 µg) was resolved by SDS-PAGE and immunoblotted with polyclonal anti-eNOS antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-iNOS antibody (Transduction Laboratories, Lexington, KY), and monoclonal anti-beta-actin (Sigma Chemical). The eNOS antibody does not cross-react with either nNOS or iNOS.

To measure relative levels of NOS gene expression using quantitative real-time PCR, we isolated total RNA from mouse lungs with the ISOGEN reagent (Nippon Gene, Tokyo, Japan). First-strand cDNA was synthesized from 1 µg of total RNA by using ExScript RT reagent kits (Takara, Otsu, Japan) and random hexamer primers. Quantitative PCR was performed using real-time SYBR Green PCR technology and an ABI PRISM 7500 Sequence Detection system (Applied Biosystems, Foster City, CA). The primers used were as described previously (20): for iNOS: forward primer, AAG GCC ACA TCG GAT TTC AC, and reverse primer, GAT GGA CCC CAA GCA AGA CTT; and for beta-actin: forward primer, CCC TAA GGC CAA CCG TGA A, and reverse primer, GTT GAA GGT CTC AAA CAT GAT CTG. Amplification reactions were performed in duplicate with SYBR Premix Ex Taq (Takara), and the thermal cycling conditions were as follows: 10 s at 95°C, 40 cycles of 5 s at 95°C, and 34 s at 60°C. Mouse iNOS and eNOS expression was normalized to beta-actin mRNA expression.

NOS activity. NOS enzymatic activity was determined as the conversion of L-[3H]arginine to L-[3H]citrulline with saturating concentrations of substrate and cofactors (Cayman Chemical, Ann Arbor, MI) as described previously (14, 22). Ca2+-dependent eNOS activity was calculated as the difference between that measured in the presence of Ca2+ and calmodulin and that measured in the presence of L-NAME, whereas the addition of 1 mM EGTA allowed the determination of the Ca2+-independent iNOS activity. Enzyme activity was expressed as L-citrulline production in picomoles per milligram of protein per minute.

Measurement of cGMP levels in mouse lung. To determine NO release from the injured lungs, we measured cyclic guanosine 3',5'-cyclic monophosphate (cGMP), an intracellular downstream indicator of NO release in BALF, using an enzyme immunoassay kit (Amersham BioScience, Piscataway, NJ) as described previously (22, 24). In brief, the right lungs were homogenized twice in ice-cold 6% trichloroacetic acid (TCA) and centrifuged at 2,000 rpm. The TCA in the supernatant fraction was extracted four times with H2O-saturated diethyl ether. The samples were then lyophilized, resuspended in assay buffer, acetylated with triethylamine/acetic anhydride, and measured with a spectrophotometer. cGMP levels are expressed as picomoles per milligram of TCA-precipitable protein solubilized with 1 N NaOH.

Statistical analysis. Statistically significant differences among groups were determined using Friedman's two-way analysis of variance for multiple comparisons between groups (StatView version 4.1; ABACUS Concepts, Berkeley, CA). The VILI scores were compared using the Mann-Whitney U-test (Microsoft Excel, Microsoft, Tokyo, Japan). Data are expressed as means ± SE, and differences are considered statistically significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lung injury induced by high VT ventilation was inhibited by eNOS overexpression. We first examined lung injury induced by high VT ventilation at various times using histopathological and cytological means. Because histopathological changes and the increased proportion of neutrophils in BALF were observed 4 h after ventilation, we used this time point for all subsequent studies.

As previously reported (22), the blood pressure of eNOS-Tg mice was lower than that of WT mice (102.0 ± 9.2 vs. 119.3 ± 9.6 mmHg, respectively; P < 0.05, n = 6–8) at the beginning of MV. Over 4 h, the blood pressure of the high VT ventilation group was maintained at a lower level (81.9 ± 11.0 mmHg, n = 6) in eNOS-Tg mice, whereas that of the low VT group did not significantly decrease (99.4 ± 9.8 mmHg, n = 8). No significant difference was evident between eNOS-Tg and WT mice during MV.

The histological examination revealed no pathological differences between WT (Fig. 1A) and eNOS-Tg mice (Fig. 1B) under normal conditions without ventilation before and after ingestion of L-NAME. Compared with normal lungs from WT and eNOS-Tg mice (Fig. 1, A and B), cell infiltration in the alveolar wall and interstitial edema were obviously increased in WT mice in the high VT ventilation groups (high VT-WT) (Fig. 1D). On the other hand, the changes were minimal in low VT ventilation groups (low VT-WT) (Fig. 1C). Interestingly, such lung inflammation induced by high VT was attenuated in eNOS-Tg mice (high VT-Tg) compared with WT mice (high VT-WT) (Fig. 1, D vs. E). Moreover, the severity of the inflammatory damage in eNOS-Tg mice given the NOS inhibitor L-NAME (high VT-Tg-L-NAME) (Fig. 1F) was similar to that found in high VT-WT mice. L-NAME did not affect the histology of WT mice in the low and high VT as well as the nonventilated groups (data not shown).


Figure 1
View larger version (81K):
[in this window]
[in a new window]
  		Fig. 1. Hematoxylin and eosin-stained lung sections. A and B: normal lungs of wild-type (WT) (A) and endothelial nitric oxide synthase-transgenic (eNOS-Tg) mice (B) without ventilation. C and D: WT mouse lungs ventilated with low tidal volume (low VT-WT; C) and high tidal volume (high VT-WT; D). E and F: Tg mouse lungs ventilated with high VT in the absence (high VT-Tg; E) or presence of N{omega}-nitro-L-arginine methyl ester (high VT-Tg-L-NAME; F). Magnification, x400. Bars, 50 µm.

 
Figure 2 shows the histopathological grades of VILI using the scoring system described in METHODS (2). The VILI scores were significantly increased in high VT-WT mice compared with WT and low VT-WT mice. In contrast, the scores in high VT-Tg mice were significantly lower than those in the high VT-WT group. Moreover, L-NAME (high VT-Tg-L-NAME) led to a higher score than that of high VT-Tg mice, whereas high VT-WT and high VT-WT-L-NAME mice did not differ. These findings suggest that eNOS overexpression in the lungs inhibits the development of VILI.


Figure 2
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 2. Semiquantitative analysis of ventilator-induced lung injury (VILI). VILI scores are based on leukocyte infiltration, exudative edema, hemorrhage, and alveolar wall thickness as described in METHODS. Values are means ± SE of at least 5 mice per group. *P < 0.0001; #P < 0.001.

 
Airway neutrophil inflammation was attenuated in eNOS-Tg mice. The BALF from untreated WT and eNOS-Tg mice contained exclusively alveolar macrophages. High VT ventilation significantly increased total cell counts in BALF from WT mice (high VT-WT) (Fig. 3A). However, the high VT ventilation-induced inflammatory cell infiltration was completely attenuated by eNOS overexpression (high VT-Tg). Treating high VT-Tg mice with L-NAME (high VT-Tg-L-NAME group) increased the total cell counts compared with high VT-Tg mice, but the increase was not statistically significant. High VT-Tg-L-NAME and high VT-WT-L-NAME mice did not differ.


Figure 3
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 3. Bronchoalveolar lavage fluid (BALF) analysis in VILI. A: total cell count (TCC). B: neutrophil cells as a ratio (%) of TCC in BALF. After mechanical ventilation (MV), BALF was obtained and examined using Diff Quik staining. Values are means ± SE of at least 5 mice per group. *P = 0.0005; #P < 0.05.

 
The high VT ventilation significantly increased neutrophil infiltration in BALF in both WT and eNOS-Tg mice (Fig. 3B). The ratio (%) of neutrophils to total cells was significantly higher in high VT-WT mice. Moreover, this neutrophil infiltration was significantly decreased in high VT-Tg mice (Fig. 3B). When the high VT-Tg mice were treated with L-NAME, however, the neutrophil proportion in high VT-Tg mice was similar to that in high VT-WT mice. These data indicate that the high VT ventilation-induced neutrophil infiltration in the airway was inhibited by eNOS overexpression.

Increases in lung edema after VILI were reduced in eNOS-Tg mice. Lung edema induced by acute lung inflammation was evaluated using the lung W/D ratio. This ratio did not significantly differ between the low VT ventilation WT and eNOS-Tg groups. However, high VT ventilation evoked severe lung edema in WT mice (high VT-WT), whereas the same procedure in eNOS-Tg mice (high VT-Tg) did not induce significant lung edema (Fig. 4). The lung W/D ratio after L-NAME treatment was not altered in high VT-WT mice but increased in high VT-Tg mice. Consequently, the water content of the lungs in high VT-Tg mice was similar to that in high VT-WT mice when the mice were treated with L-NAME. These data indicate that the lung edema induced by high VT ventilation was inhibited by eNOS overexpression.


Figure 4
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 4. Lung water content after VILI. The ratio of wet to dry weight (W/D) of lungs was determined in right whole lungs by dividing wet weight by dry weight. Values are means ± SE of at least 6 mice per group. *P < 0.0001.

 
Lung eNOS expression and activity were increased in eNOS-Tg mice. We previously localized eNOS overexpression in pulmonary airway and alveolar cells as well as in the endothelium of pulmonary arteries and veins of eNOS-Tg mice (22). Consistent with these results, eNOS protein levels in the lungs of eNOS-Tg mice were significantly higher than those in WT mice (Fig. 5A). Notably, high VT ventilation did not change the eNOS protein level in the lungs of WT and eNOS-Tg mice. Similarly, the eNOS levels were not increased in mice given L-NAME, regardless of ventilation. On the other hand, the amount of iNOS protein was not significantly increased in this model even after MV, and quantitative PCR confirmed that iNOS mRNA was not induced by the MV, in contrast to the marked iNOS induction by LPS (Fig. 5B).


Figure 5
View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5. Expression and activity of eNOS and inducible NOS (iNOS) in mouse lungs. A: Western blots were used to detect eNOS, iNOS, and beta-actin protein in lung homogenates. B: quantitative PCR was used to determine mRNA levels of iNOS. C: conversion of L-[3H]arginine to L-[3H]citrulline was used to determine NOS activity, shown as Ca2+-dependent (eNOS, open bars) and -independent activity (iNOS, solid bars). D: cGMP levels in lung homogenates were determined using enzyme immunoassay. Values are means ± SE of 5 mice per group. *P < 0.005; #P < 0.05.

 
We then examined NOS activity as the conversion assay of L-arginine to L-citrulline (Fig. 5C). Ca2+-dependent eNOS activity was easily detected in eNOS-Tg mice and was inhibited by L-NAME. In contrast, Ca2+-independent iNOS activity was not significantly increased by the ventilation procedures in either mouse group. Similarly, cGMP analysis revealed that eNOS-Tg mice had two- to threefold higher basal cGMP levels than WT mice, and the MV procedure did not augment the cGMP accumulation (Fig. 5D). These findings suggest that our MV procedure did not stimulate iNOS induction in mice.

Cytokine levels in plasma and BALF were reduced in eNOS-Tg mice. Injurious ventilatory strategies increase the concentrations of various inflammatory cytokines (30), and among these, we measured IL-6 and TNF-{alpha} levels as markers of inflammation (Fig. 6). Both TNF-{alpha} and IL-6 levels were increased by high VT ventilation in BALF and plasma, although IL-6 values in BALF did not significantly differ between WT and eNOS-Tg mice. On the other hand, the increased TNF-{alpha} level in the plasma of high VT-WT mice (446.5 ± 174.7 pg/ml, P < 0.001 vs. WT) was significantly reduced in high VT-Tg mice (59.2 ± 12.8 pg/ml, P < 0.0001 vs. high VT-WT), and the finding was similar in BALF. L-NAME (high VT-Tg-L-NAME) slightly increased the plasma level of IL-6 and TNF-{alpha} compared with the high VT-Tg group, whereas no significant change was evident in WT mice given L-NAME (high VT-WT-L-NAME). That is, IL-6 and TNF-{alpha} levels in plasma and BALF did not significantly differ between WT and eNOS-Tg mice in the presence of L-NAME.


Figure 6
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 6. Cytokine levels in plasma (A and B) and BALF (C and D) measured using ELISA after MV. Both cytokine levels in plasma were lower in high VT-Tg than in high VT-WT mice. BALF levels of IL-6 (C) were not affected by eNOS overexpression, whereas those of TNF-{alpha} (D) were decreased in high VT-Tg mice (n = 6–8 mice per group). Values are means ± SE of five mice per group. *P < 0.005; #P < 0.05.

 
Figures 7A and 8A show that MIP-2 concentrations were significantly increased in high VT-WT mice (326.1 ± 84.9 pg/ml in BALF and 1,808.5 ± 320.9 pg/ml in plasma) compared with in WT mice (P < 0.005). These increases were appreciably attenuated in high VT-Tg mice (59.1 ± 14.7 pg/ml in BALF and 178.9 ± 80.0 pg/ml in plasma) compared with high VT-WT (P < 0.001). This attenuation was abolished by L-NAME only in eNOS-Tg mice (314.9 ± 80.1 pg/ml in BALF and 1,479.0 ± 326.8 pg/ml in plasma). Furthermore, the plasma concentration profile of MCP-1 (Fig. 8B) was similar to that of MIP-2. Under high VT ventilation, the plasma level of MCP-1 was significantly increased in high VT-WT mice (2,252.7 ± 527.8 pg/ml, P < 0.001 vs. WT) and attenuated in high VT-Tg mice (254.9 ± 82.1 pg/ml, P < 0.001 vs. high VT-WT). On the other hand, BALF levels of MCP-1 were increased in high VT-WT mice, although the difference was not statistically significant (P = 0.099 vs. WT, Fig. 7B).


Figure 7
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 7. Macrophage inflammatory protein (MIP)-2 and macrophage chemoattractant protein (MCP)-1 concentrations in BALF. Release of MIP-2 (A) and MCP-1 (B) into BALF after MV was evaluated using ELISA (n = 6–8 mice per group). The concentration of MIP-2 was increased by MV (high VT-WT) and obviously reduced in high VT-Tg. The concentration of MCP-1 tended to increase similarly to MIP-2, but the difference was not statistically significant. Values are means ± SE of 5 mice per group. *P < 0.01.

 

Figure 8
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 8. MIP-2 and MCP-1 concentrations in plasma. Release of MIP-2 (A) and MCP-1 (B) into plasma after MV was measured using ELISA (n = 6–8 mice per group). Concentrations of MIP-2 and MCP-1 were significantly elevated in high VT-WT mice and obviously attenuated in high VT-Tg mice. Values are means ± SE of 5 mice per group. *P < 0.001; #P < 0.005.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that MV with a high VT caused lung edema and inflammation in mice, which was accompanied by cytokine/chemokine release. In contrast, a low VT caused only minimal histological damage to the lungs. These tidal volume-dependent lung injuries in humans have been documented, where the low and high VT is related to the protective and conventional strategy, respectively, in agreement with the clinical study reported by the ARDS network (1). Our VILI scoring system confirmed that the histopathological grading of VILI closely matched the degree of tidal volume.

One of the major findings of this study was that the high VT-induced lung injury was inhibited in eNOS-Tg mice. Although it is widely accepted that NO modulates inflammation through a variety of mechanisms (15, 19) and NO is produced by several NOS isoforms, the relative contribution of these isoforms in inflammation has not been defined. Several animal models have been established to characterize the role of NOS in lung inflammation (6, 11, 14, 21). For instance, chemicals such as LPS evoke lung injury, but defining the role of eNOS in the chemically induced lung injury model is difficult, because endotoxin exposure directly activates iNOS. In the present study, we found that iNOS levels were not affected by the MV procedure in mice, which is consistent with the results of a previous study showing that that MV does not induce iNOS expression in rats (6). The NOS activity assay confirmed that the MV procedure did not stimulate Ca2+-independent iNOS activity. Furthermore, the effect of eNOS overexpression was reversed by L-NAME. From these results, we concluded that the reduction of VILI in eNOS-Tg mice was mainly caused by NO derived from overexpressed eNOS in the lung.

On the other hand, a recent study by Peng et al. (25) showed that iNOS may be an important contributor to VILI and that iNOS protein was increased after 2-h MV. We could not resolve the discrepancy of iNOS expression in VILI; however, there are many methodological differences in the ventilation protocol as described in the article. We speculate that our blood pressure monitoring without a cannulation procedure might cause some influence on the data, and our present study is similar to that described by Hammerschmidt et al. (9) in which high VT ventilation reduced eNOS gene expression but did not affect iNOS expression in isolated rabbit lungs.

It has been postulated that eNOS-derived NO plays a protective role against lung injury. MacRitchie et al. (21) showed that decreased eNOS protein expression in both pulmonary artery and airways is associated with lung injury in preterm lambs ventilated for 3 wk. Furthermore, eNOS-deficient mice are susceptible to severe lung edema and inflammation after ischemia-reperfusion injury (11). These findings, together with our present data, have strengthened the notion that NO derived from chronic eNOS overexpression may protect the lung from VILI.

Several plausible mechanisms could explain the reduced VILI in eNOS-Tg mice. First, NO derived from eNOS could inhibit neutrophil infiltration and macrophage activation. Activated macrophages respond immediately to the overdistension of alveolar tissue, attract neutrophils by releasing proinflammatory cytokines, and play an important role in initiating the inflammation associated with VILI (10). Kawano et al. (12) found that the diffuse infiltration by activated neutrophils was induced by MV in the rabbit lung, whereas lung damage was minimal in neutrophil-depleted animals. Our observation also supports the evidence that neutrophils are one of the key regulators during the pathogenesis of VILI.

Second, NO could alter cytokine/chemokine release and receptor expression in the lung. Proinflammatory cytokines and chemokines are produced during injurious ventilation and ALI/ARDS (30). For instance, the CXC chemokines (MIP-2, KC) attract neutrophils into the lung, whereas CC chemokines (MCP-1, MCP-3) attract lymphocytes and monocytes and activate macrophages (23). Belperio et al. (2) demonstrated that KC/CXCL1 and MIP-2/CXCL2/3, through interaction with their shared receptor, CXC chemokine receptor 2 (CXCR2), regulates neutrophil recruitment by promoting neutrophil adherence to endothelial cells and transendothelial migration into lung tissue. On the other hand, cell surface expression of CXCR2 is increased on nonleukocyte cell populations in the high VT group (31). These findings imply that mechanical stimulation of nonleukocytes upregulates chemokine receptor expression by fibroblasts as well as by epithelial and endothelial cells, which play a significant role in mediating VILI via neutrophil recruitment.

The present study found that both TNF-{alpha} and IL-6 levels were increased by high VT ventilation in BALF and plasma and that the expression was decreased in eNOS-Tg mice. The concentration of TNF-{alpha} in BALF was similar to that of plasma, but the change in TNF-{alpha} was more significant in plasma. These data suggest that VILI caused systemic injury and imply that plasma TNF-{alpha} levels may be more sensitive to lung injury. The overexpression of eNOS also reduced MIP-2 release after MV. Moreover, MCP-1 levels were decreased in high VT-Tg compared with high-WT mice, especially in plasma. These molecules are important mediators of lung inflammation by interaction with NO (3, 29, 32, 36). On the other hand, NO blocks the upregulation of adhesion molecules such as VCAM-1 (7), ICAM-1, and P-selectin (17). We did not show evidence that our VILI model affects these adhesion molecule expressions in this study; however, these facts suggested that the inhibition of inflammatory cytokines and adhesion molecules by overproduction of NO might play some roles in hematopoietic-endothelial cell interaction in the pathogenesis of VILI.

The anti-inflammatory effect of NO might be useful in a therapeutic environment. Inhaled NO reduces lung inflammation and injury in premature lambs that receive MV for 3 h after birth (13). Inhaled NO is now used clinically to treat acute lung injury in humans (31), although a beneficial effect on survival has not yet been established. Our study found that eNOS was chronically overexpressed mainly in vascular endothelial cells, and we showed that NO derived from endothelial cells inhibited neutrophil-associated lung edema and inflammation by reducing cytokine/chemokine production in VILI. These data support previous findings indicating that NO may help to prevent VILI. However, the clinical effect and approach of eNOS delivery have not been established and should be evaluated by further studies.


   ACKNOWLEDGMENTS
 
We thank Dr. Chiho Obayashi (Department of Pathology, Kobe University Graduate School of Medicine) for helpful advice during the histopathological analyses.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Y. Nishimura, Division of Cardiovascular and Respiratory Medicine, Kobe Univ. Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan (e-mail: nishiy{at}med.kobe-u.ac.jp)

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
 REFERENCES
 

  1. The Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volume as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 342: 1301–1308, 2000.[Abstract/Free Full Text]
  2. Belperio JA, Keane MP, Burdick MD, Londhe V, Xue YY, Li K, Phillips RJ, and Strieter RM. Critical role for CXCR2 and CXCR2 ligands during the pathogenesis of ventilator-induced lung injury. J Clin Invest 110: 1703–1716, 2002.[CrossRef][Web of Science][Medline]
  3. von Bethmann AN, Brasch F, Nusing R, Vogt K, Volk HD, Muller KM, Wendel A, and Uhlig S. Hyperventilation induces release of cytokines from perfused mouse lung. Am J Respir Crit Care Med 157: 263–272, 1998.
  4. Bogdan C. Nitric oxide and the immune response. Nat Immunol 2: 907–916, 2001.[CrossRef][Web of Science][Medline]
  5. Chiumello D, Pristine G, and Slutsky AS. Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome. Am J Respir Crit Care Med 160: 109–116, 1999.[Abstract/Free Full Text]
  6. Choi WI, Quinn DA, Park KM, Moufarrej RK, Jafari B, Syrkina O, Bonventre JV, and Hales CA. Systemic microvascular leak in an in vivo rat model of ventilator-induced lung injury. Am J Respir Crit Care Med 167: 1627–1632, 2003.[Abstract/Free Full Text]
  7. De Caterina R, Libby P, Peng HB, Thannickal VJ, Rajavashisth TB, Gimbrone MA Jr, Shin WS, and Liao JK. Nitric oxide decreases cytokine-induced endothelial activation: nitric oxide selectively reduces endothelial expression of adhesion molecules and proinflammatory cytokines. J Clin Invest 96: 60–68, 1995.[Web of Science][Medline]
  8. Dreyfuss D and Saumon G. Ventilator-induced lung injury: lessons from experimental studies. Am J Respir Crit Care Med 157: 294–323, 1998.
  9. Hammerschmidt S, Schiller J, Kuhn H, Meybaum M, Gessner C, Sandvoss T, Arnold K, and Wirtz H. Influence of tidal volume on pulmonary NO release, tissue lipid peroxidation and surfactant phospholipids. Biochim Biophys Acta 1639: 17–26, 2003.[Medline]
  10. Imanaka H, Shimaoka M, Matsuura N, Nishimura M, Ohta N, and Kiyono H. Ventilator-induced lung injury is associated with neutrophil infiltration, macrophage activation, and TGF-beta1 mRNA upregulation in rat lungs. Anesth Analg 92: 428–436, 2001.[Abstract/Free Full Text]
  11. Kaminski A, Pohl CB, Sponholz C, Ma N, Stamm C, Vollmar B, and Steinhoff G. Up-regulation of endothelial nitric oxide synthase inhibits pulmonary leukocyte migration following lung ischemia-reperfusion in mice. Am J Pathol 164: 2241–2249, 2004.[Abstract/Free Full Text]
  12. Kawano T, Mori S, Cybulsky M, Burger R, Ballin A, Cutz E, and Bryan AC. Effect of granulocyte depletion in a ventilated surfactant-depleted lung. J Appl Physiol 62: 27–33, 1987.[Abstract/Free Full Text]
  13. Kinsella JP, Parker TA, Galan H, Sheridan BC, Halbower AC, and Abman SH. Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease. Pediatr Res 41: 457–463, 1997.[Web of Science][Medline]
  14. Kristof AS, Goldberg P, Laubach V, and Hussain SN. Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury. Am J Respir Crit Care Med 158: 1883–1889, 1998.[Abstract/Free Full Text]
  15. Kubes P, Suzuki M, and Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 88: 4651–4655, 1991.[Abstract/Free Full Text]
  16. Kubes P and Granger DN. Nitric oxide modulates microvascular permeability. Am J Physiol Heart Circ Physiol 262: H611–H615, 1992.[Abstract/Free Full Text]
  17. Kubes P, Kurose I, and Granger DN. NO donors prevent integrin-induced leukocyte adhesion but not P-selectin-dependent rolling in postischemic venules. Am J Physiol Heart Circ Physiol 267: H931–H937, 1994.[Abstract/Free Full Text]
  18. Kubes P. Nitric oxide affects microvascular permeability in the intact and inflamed vasculature. Microcirculation 2: 235–244, 1995.[Medline]
  19. Kurose I, Kubes P, Wolf R, Anderson DC, Paulson J, Miyasaka M, and Granger DN. Inhibition of nitric oxide production: mechanisms of vascular albumin leakage. Circ Res 73: 164–171, 1993.[Abstract]
  20. Lee JK, Borhani M, Ennis TL, Upchurch GR Jr, Thompson RW. Experimental abdominal aortic aneurysms in mice lacking expression of inducible nitric oxide synthase. Arterioscler Thromb Vasc Biol 21: 1393–401, 2001.[Abstract/Free Full Text]
  21. MacRitchie AN, Albertine KH, Sun J, Lei PS, Jensen SC, Freestone AA, Clair PM, Dahl MJ, Godfrey EA, Carlton DP, and Bland RD. Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs. Am J Physiol Lung Cell Mol Physiol 281: L1011–L1020, 2001.[Abstract/Free Full Text]
  22. Ohashi Y, Kawashima S, Hirata K, Yamashita T, Ishida T, Inoue N, Sakoda T, Kurihara H, Yazaki Y, and Yokoyama M. Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase. J Clin Invest 102: 2061–2071, 1998.[Web of Science][Medline]
  23. Olson TS and Ley K. Chemokines and chemokine receptors in leukocyte trafficking. Am J Physiol Regul Integr Comp Physiol 283: R7–R28, 2002.[Abstract/Free Full Text]
  24. Ozaki M, Kawashima S, Yamashita T, Ohashi Y, Rikitake Y, Inoue N, Hirata K, Hayashi Y, Itoh H, and Yokoyama M. Reduced hypoxic pulmonary vascular remodeling by nitric oxide from the endothelium. Hypertension 37: 322–327, 2001.[Abstract/Free Full Text]
  25. Peng X, Abdulnour RE, Sammani S, Ma SF, Han EJ, Hasan EJ, Tuder R, Garcia JG, and Hassoun PM. Inducible nitric oxide synthase contributes to ventilator-induced lung injury. Am J Respir Crit Care Med 172: 470–479, 2005.[Abstract/Free Full Text]
  26. Pinhu L, Whitehead T, Evans T, and Griffiths M. Ventilator-associated lung injury. Lancet 361: 332–340, 2003.[CrossRef][Web of Science][Medline]
  27. Quinn DA, Moufarrej RK, Volokhov A, and Hales CA. Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury. J Appl Physiol 93: 517–525, 2002.[Abstract/Free Full Text]
  28. Skidgel RA, Gao XP, Brovkovych V, Rahman A, Jho D, Predescu S, Standiford TJ, and Malik AB. Nitric oxide stimulates macrophage inflammatory protein-2 expression in sepsis. J Immunol 169: 2093–2101, 2002.[Abstract/Free Full Text]
  29. Sokol J, Jacobs SE, and Bohn D. Inhaled nitric oxide for acute hypoxic respiratory failure in children and adults: a meta-analysis. Anesth Analg 97: 989–998, 2003.[Abstract/Free Full Text]
  30. Tremblay L, Valenza F, Ribeiro SP, Li J, and Slutsky AS. Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 99: 944–952, 1997.[Web of Science][Medline]
  31. Tremblay LN, Miatto D, Hamid Q, Govindarajan A, and Slutsky AS. Injurious ventilation induces widespread pulmonary epithelial expression of tumor necrosis factor-{alpha} and interleukin-6 messenger RNA. Crit Care Med 30: 1693–1700, 2002.[CrossRef][Web of Science][Medline]
  32. Uhlig S. Ventilation-induced lung injury and mechanotransduction: stretching it too far? Am J Physiol Lung Cell Mol Physiol 282: L892–L896, 2002.[Abstract/Free Full Text]
  33. Ware LB and Matthay MA. The acute respiratory distress syndrome. N Engl J Med 342: 1334–1349, 2000.[Free Full Text]
  34. Wilson MR, Choudhury S, Goddard ME, O'Dea KP, Nicholson AG, and Tanaka M. High tidal volume ventilation upregulates intrapulmonary cytokines in an in vivo mouse model of ventilator-induced lung injury. J Appl Physiol 95: 1385–1393, 2003.[Abstract/Free Full Text]
  35. Yamashita T, Kawashima S, Ohashi Y, Ozaki M, Ueyama T, Ishida T, Inoue N, Hirata K, Akita H, and Yokoyama M. Resistance to endotoxin shock in transgenic mice overexpressing endothelial nitric oxide synthase. Circulation 101: 931–937, 2000.[Abstract/Free Full Text]
  36. Zeiher AM, Fisslthaler B, Schray-Utz B, and Busse R. Nitric oxide modulates the expression of monocyte chemoattractant protein 1 in cultured human endothelial cells. Circ Res 76: 980–986, 1995.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
E. P. Schmidt, M. Damarla, O. Rentsendorj, L. E. Servinsky, B. Zhu, A. Moldobaeva, A. Gonzalez, P. M. Hassoun, and D. B. Pearse
Soluble guanylyl cyclase contributes to ventilator-induced lung injury in mice
Am J Physiol Lung Cell Mol Physiol, December 1, 2008; 295(6): L1056 - L1065.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
R. A. Oeckler and R. D. Hubmayr
Ventilator-associated lung injury: a search for better therapeutic targets
Eur. Respir. J., December 1, 2007; 30(6): 1216 - 1226.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. Liu, Y. Hotta, A. R. Graveline, O. V. Evgenov, E. S. Buys, K. D. Bloch, F. Ichinose, and W. M. Zapol
Congenital NOS2 deficiency prevents impairment of hypoxic pulmonary vasoconstriction in murine ventilator-induced lung injury
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1300 - L1305.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
T. Miyahara, K. Hamanaka, D. S. Weber, D. A. Drake, M. Anghelescu, and J. C. Parker
Phosphoinositide 3-kinase, Src, and Akt modulate acute ventilation-induced vascular permeability increases in mouse lungs
Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L11 - L21.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
S. Vyas-Read, P. W. Shaul, I. S. Yuhanna, and B. C. Willis
Nitric oxide attenuates epithelial-mesenchymal transition in alveolar epithelial cells
Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L212 - L221.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/6/L1078    most recent
00239.2005v1
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 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 Web of Science (14)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Takenaka, K.
Right arrow Articles by Yokoyama, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Takenaka, K.
Right arrow Articles by Yokoyama, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.