Nitric oxide synthase 3 contributes to ventilator-induced lung injury

Katerina Vaporidi, Roland C. Francis, Kenneth D. Bloch and Warren M. Zapol

Am J Physiol Lung Cell Mol Physiol 299:L150-L159, 2010. First published 7 May 2010; doi:10.1152/ajplung.00341.2009 You might find this additional info useful... Supplemental material for this article can be found at: http://ajplung.physiology.org/content/suppl/2010/05/17/ajplung.00341.2009.DC1.html This article cites 42 articles, 23 of which can be accessed free at: http://ajplung.physiology.org/content/299/2/L150.full.html#ref-list-1 This article has been cited by 1 other HighWire hosted articles Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury Dejie Liu, Zhibo Yan, Richard D. Minshall, David E. Schwartz, Yuguo Chen and Guochang Hu Am J Physiol Lung Cell Mol Physiol, February 15, 2012; 302 (4): L370-L379. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://ajplung.physiology.org/content/299/2/L150.full.html Additional material and information about AJP - Lung Cellular and Molecular Physiology can be found at: http://www.the-aps.org/publications/ajplung

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AJP - Lung Cellular and Molecular Physiology publishes original research covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and components of the respiratory system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2010 by the American Physiological Society. ISSN: 1040-0605, ESSN: 1522-1504. Visit our website at http://www.the-aps.org/.

Am J Physiol Lung Cell Mol Physiol 299: L150L159, 2010. First published May 7, 2010; doi:10.1152/ajplung.00341.2009.

Nitric oxide synthase 3 contributes to ventilator-induced lung injury

Katerina Vaporidi, Roland C. Francis, Kenneth D. Bloch, and Warren M. Zapol
Anesthesia Center for Critical Care Research, Department of Anesthesia and Critical Care, Massachusetts General Hospital and Harvard Medical School, Boston Massachusetts
Submitted 25 September 2009; accepted in nal form 6 May 2010

Vaporidi K, Francis RC, Bloch KD, Zapol WM. Nitric oxide synthase 3 contributes to ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 299: L150L159, 2010. First published May 7, 2010; doi:10.1152/ajplung.00341.2009.Nitric oxide synthase (NOS) depletion or inhibition reduces ventilator-induced lung injury (VILI), but the responsible mechanisms remain incompletely dened. The aim of this study was to elucidate the role of endothelial NOS, NOS3, in the pathogenesis of VILI in an in vivo mouse model. Wild-type and NOS3-decient mice were ventilated with high-tidal volume (HVT; 40 ml/kg) for 4 h, with and without adding NO to the inhaled gas. Additional wild-type mice were pretreated with tetrahydrobiopterin and ascorbic acid, agents that can prevent NOS-generated superoxide production. Arterial blood gas tensions, histology, and lung mechanics were evaluated after 4 h of HVT ventilation. The concentration of protein, IgM, cytokines, malondialdehyde, and 8-isoprostane were measured in bronchoalveolar lavage uid (BALF). Myeloperoxidase activity, total and oxidized glutathione levels, and NOS-derived superoxide production were measured in lung tissue homogenates. HVT ventilation induced VILI in wild-type mice, as reected by decreased lung compliance, increased concentrations of protein and cytokines in BALF, and oxidative stress. All indices of VILI were ameliorated in NOS3-decient mice. Augmenting pulmonary NO levels by breathing NO during mechanical ventilation did not increase lung injury in NOS3-decient mice. HVT ventilation increased NOS-inhibitable superoxide production in lung extracts from wild-type mice but not in those from NOS3-decient mice. Administration of tetrahydrobiopterin and ascorbic acid ameliorated VILI in wild-type mice. Our results indicate that NOS3 contributes to ventilator-induced lung injury via increased production of superoxide. superoxide; pulmonary edema; inammation
MECHANICAL VENTILATION

as substrate (L-arginine) or cofactor (e.g., tetrahydrobiopterin, BH4) depletion, NOS3 catalyzes the uncoupled reduction of O2 and produces superoxide (13). Under conditions of increased oxidative stress, such as those known to develop in VILI (8, 29), BH4 levels may decline leading to NOS3 uncoupling (22, 23, 26). ROS production by uncoupled NOS3 contributes to endothelial dysfunction observed in hypertension, hyperglycemia, and hypercholesterolemia (3, 13, 23, 24). NOS3 uncoupling, ROS production, and endothelial dysfunction can be prevented by administration of BH4 and ascorbic acid (22, 27). It is known that ROS can disrupt the endothelial barrier and induce inammation (8, 9, 16, 40). The role of ROS in the pathogenesis of VILI is generally accepted (8, 29), but the sources of ROS have not been fully dened. In the present study, we hypothesized that NOS3 contributes to the pathogenesis of VILI, not by producing NO, but rather by generating superoxide. To test this hypothesis, we compared the effect of HVT ventilation in wild-type (WT) and NOS3/ mice. We replenished NO in NOS3/ mice by adding NO to the inhaled gas during mechanical ventilation. We evaluated oxidative stress and NOS-derived superoxide production in lungs of WT and NOS3/ mice subjected to HVT ventilation. Finally, we examined the effect of BH4 and ascorbic acid, which can prevent NOS3 uncoupling, in WT mice subjected to HVT ventilation.
METHODS

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with high-tidal volumes (HVT) may initiate and/or exacerbate acute lung injury (ALI) via a process known as ventilator-induced lung injury (VILI) (14, 29). Shear forces generated during HVT ventilation induce alveolar-capillary barrier disruption and pulmonary inammation. Several mechanisms have been implicated in the development of VILI, including neutrophil inltration, as well as increased production of proinammatory cytokines, reactive oxygen species (ROS), and nitric oxide (NO) (14, 29, 33). NO is produced by three NO synthase (NOS) isoforms: neuronal, inducible, and endothelial NOS (NOS 1, 2, and 3, respectively) (12). Several studies have shown that NOS2 contributes to VILI (15, 25, 30), whereas the role of NOS3 remains controversial. Transgenic mice overexpressing NOS3 (38) and mice that were congenitally NOS3 decient (36) were both found to be protected from VILI. NOS3 couples L-arginine oxidation and O2 reduction leading to the production of NO. Under certain conditions, such

Address for reprint requests and other correspondence: W. M. Zapol, Dept. of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, 55 Fruit St., Boston, MA 02114 (e-mail: wzapol@partners.org). L150

Mouse model of VILI. Male C57BL/6 mice (WT) and NOS3/ mice at 7 8 wk of age were studied as described in detail in the online data supplement (Supplemental data for this article is available at the AJP-Lung web site). The study was approved by the Subcommittee for Research Animal Care of the Massachusetts General Hospital. Briey, after induction of anesthesia, a tracheostomy was performed, and mice were connected to a ventilator (Inspira; Harvard Apparatus, Boston, MA). Mice were ventilated on volume control mode, initially at a tidal volume (VT) of 8 ml/kg and respiratory rate (RR) 125 breaths/min for 1 h, to permit hemodynamic stabilization. The carotid artery was catheterized for blood pressure monitoring, uid and anesthetic administration, and blood sampling. After 1 h, VT was increased to 40 ml/kg, and RR decreased to 60 breaths/min, at a PEEP of 1 cmH2O and FIO2 of 0.5 (maintained constant throughout the study). After 4 h of HVT ventilation, blood was collected, and an inspiratory pressure-volume curve of the respiratory system was obtained by slow ination of the lungs, as previously described (34). Subsequently, bronchoalveolar lavage was performed, and the lungs were collected for further analysis. As controls, additional animals of both genotypes were anesthetized and ventilated briey (1 min) until paralyzed, with a VT of 8 ml/kg and RR of 125 breaths/min, whereupon an inspiratory pressure volume curve was obtained, followed by bronchoalveolar lavage and tissue collection. For histological evaluation, lungs from mice not subjected to BALF collection were inated with 4% paraformaldehyde at a transpulmonary pressure of 25 cmH2O. A detailed description of the experimental procedure is provided in the online data supplement and in Supplementary Fig. S1.
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The tidal volume and the duration of ventilation were chosen based on results from pilot experiments, which revealed that these conditions caused VILI in WT mice with a reproducible alteration in lung mechanics and inammation. Experimental groups. We studied nine groups of mice. We studied WT and NOS3/ mice under control conditions and after 4 h of HVT ventilation. In addition, we studied WT and NOS3/ mice subjected to HVT ventilation while receiving inhaled NO, at either a concentration of 5 or 50 parts per million (ppm; MedicalTechnical Gases, Medford, MA) added to inspiratory gas from the initiation of mechanical ventilation. Finally, we studied a group of WT mice that was treated with BH4 (Schricks Laboratory, Switzerland) dissolved in an ascorbic acid solution (Bioniche Pharma, Lake Forest, IL; 100 mg/kg of both BH4 and ascorbic acid, injected ip) before the initiation of HVT ventilation. The number of animals in each experimental group is presented in Table 1. The number of animals studied in each group was based on our pilot studies that revealed at least ve mice in each group were required to show a difference in VILI between WT and NOS3/ mice subjected to HVT ventilation. Additional mice were included to provide adequate samples for biochemical and histological studies and to conrm the stability of the model over time. Evaluation of lung injury and inammation. Protein concentration in BALF was measured with a bicinchoninic acid assay (Pierce Chemical, Rockford, IL). IgM levels in BALF were measured with an ELISA (Genway Biotech). The levels of the proinammatory cytokines, IL-6, TNF, and macrophage inammatory protein-2 (MIP-2), were measured in BALF and plasma using ELISAs (R&D Systems, Minneapolis, MN). Total and differential cell counts in BALF and lung myeloperoxidase (MPO) activity were performed as described in the online data supplement. Because sample quantities from individual animals were insufcient to perform all assays, a randomly chosen subset of samples was used for each assay. The number of samples used in each assay is reported in the gure legends. Parafn-embedded lung sections were sectioned 6 m thick and stained with hematoxylin and eosin for histology. Staining for neutrophils was performed in the same lung sections using antimouse neutrophil monoclonal antibody (CL8993 AP; Cedarline Laboratories, Ontario, Canada). Measurement of nitric oxide metabolites and NO synthase gene expression. Total nitrite/nitrate levels in BALF were measured using a uorimetric assay (Cayman Chemical). Methods for measurement of pulmonary NOS1, NOS2, and NOS3 mRNA levels are described in the online data supplement. Evaluation of oxidative stress and ROS production. Malondialdehyde (MDA) levels in BALF were measured using a spectrophotometric assay for MDA (Oxis International, Foster City, CA). BALF 8-isoprostane levels were measured using an EIA assay for 8-isoprostane (Cayman Chemical). Total and oxidized glutathione levels in lung tissue were measured using an enzymatic assay (Cayman Chemical). Superoxide production was measured in lung homogenates using a lucigenin-enhanced chemiluminescence assay supplemented with

-nicotinamide adenine dinucleotide 2=-phosphate reduced tetrasodium salt (NADPH) in the presence and absence of nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor, as described in the online data supplement. Statistical analysis. Data were compared by two-way ANOVA (the non-parametric tests Mann-Whitney rank sum test and Kruskal-Wallis were used when normality and equal variance test failed). When the P value was less than 0.05, the Bonferroni post hoc test was applied. All analyses were performed using SigmaStat statistical software. All data in text and tables are expressed as means SD, and data are presented in gures as box plots. Signicance was dened as P 0.05.
RESULTS

Table 1. Experimental groups and number of animals in each group

Treatment Control HVT HVTiNO5ppm HVTiNO50ppm HVTBH4/asc

Genotype WT NOS3/

11 11

19 19

10 10

7 7

Number of animals in each of 9 study groups: control mice; mice subjected to 4 h of high-tidal volume (HVT) ventilation (VT 40 ml/kg) without (HVT) or with 5 ppm (HVTiNO5ppm) or 50 ppm (HVTiNO50ppm) inhaled NO; and WT mice treated with BH4 and ascorbic acid (HVTBH4/asc). AJP-Lung Cell Mol Physiol VOL

Effects of HVT ventilation on WT and NOS3/ mice. In all mice, peak inspiratory pressure (PIP) at the initiation of HVT (40 ml/kg) was 36 0.5 cmH2O. Shortly after the initiation of HVT ventilation, PIP decreased modestly in all mice, possibly due to resolution of atelectasis. In WT mice, PIP increased after 2 h of HVT ventilation and was 38.8 2.2 cmH2O after 4 h (P 0.001 vs. PIP at initiation of HVT; Fig. 1A). In NOS3/ mice, PIP was not different at the initiation and at the end of 4 h of HVT ventilation. The pressure-volume curve of the respiratory system was not different in control WT and NOS3/ mice. HVT ventilation for 4 h decreased lung compliance in WT and NOS3/ mice, as reected by the downward shift of the pressure-volume curve of the respiratory system (Fig. 1B). However, after 4 h of HVT, lung compliance was higher in NOS3/ mice than in WT mice. In WT mice, the development of pulmonary edema, as indicated by the decline in lung compliance, was associated with impaired oxygenation, as reected by a decreased PaO2 and an increased alveolar-arterial oxygen difference [D(Aa)O2, Table 2]. After HVT ventilation, D(A-a)O2 was less in NOS3/ mice than in WT mice (34 13 vs. 137 91 mmHg, P 0.001). There were no differences in PaCO2, pH, and HCO 3 between the two genotypes after 4 h of HVT ventilation (Table 2). Concentrations of protein and IgM in BALF did not differ in control WT and NOS3/ mice. BALF protein concentrations were greater in WT and NOS3/ mice ventilated with HVT than in corresponding control mice consistent with increased alveolar-capillary permeability (Fig. 2). Moreover, IgM, which, due to its high-molecular-weight (900 kDa), is largely excluded from the BALF under normal conditions (19), was increased in the BALF of WT and NOS3/ mice exposed to HVT ventilation (Fig. 3). However, BALF total protein and IgM concentrations were lower in NOS3/ mice exposed to HVT ventilation than in similarly ventilated WT mice (Figs. 2 and 3). Concentrations of proinammatory cytokines in BALF and plasma, as well as leukocyte numbers in BALF and lung tissue, did not differ between control WT and NOS3/ mice. HVT ventilation induced an inammatory response in the lungs of WT and NOS3/ mice, as indicated by increased levels of IL-6, TNF, and MIP-2 in BALF (Figs. 4 and 5). HVT ventilation was also associated with increased plasma IL-6 levels in both genotypes (Fig. 5C). TNF and MIP-2 were not detected in the plasma of WT or NOS3/ mice after HVT ventilation. BALF concentrations of all measured proinammatory cytokines were less in NOS3/ mice exposed to HVT
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Fig. 1. A: peak inspiratory pressure (PIP) during high-tidal volume (HVT) ventilation (VT 40 ml/kg) in wild-type (WT) mice (gray) and NOS3/ mice (white), without (circles) or with 5 ppm (diamonds) or 50 ppm (triangles) inhaled NO, and BH4/ascorbic acid-treated WT mice (squares). During the rst 60 min of mechanical ventilation at tidal volume (VT) 8 ml/kg, PIP was 8 9 cmH2O in all mice. PIP increased during HVT ventilation in WT mice not receiving inhaled NO and in WT mice receiving 50 ppm inhaled NO (*P 0.05 vs. all other groups). For clarity, PIP only during HVT ventilation is shown. B: inspiratory pressure-volume curve of the respiratory system in WT mice (gray) and NOS3/ mice (white), in control mice (plus symbol) and in mice after HVT ventilation without (circles), or with 5 ppm (diamonds) or 50 ppm (triangles) inhaled NO, and WT mice treated with BH4 and ascorbic acid (squares). HVT ventilation resulted in deterioration of lung mechanics in all ventilated mice (P 0.001 WT-HVT vs. WT control, P 0.01 NOS3/-HVT vs. NOS3/ control). Lung compliance decreased to a greater extent in WT mice than in NOS3/ mice (P 0.01 NOS3/-HVT vs. WT-HVT). Inhaled NO at 5 or 50 ppm did not affect lung mechanics in NOS3/ mice subjected to HVT. In WT mice, breathing 5 ppm NO improved lung mechanics (P 0.01 WTHVTiNO5ppm vs. WT-HVT), whereas breathing 50 ppm NO did not. Treatment of WT mice exposed to HVT with BH4 and ascorbic acid improved lung mechanics (P 0.01 WT-HVTBH4/asc vs. WT-HVT). For clarity, only mean values are shown. N 510 per group.

ventilation than in similarly ventilated WT mice. Moreover, plasma IL-6 concentrations were less in NOS3/ than in WT mice after HVT ventilation. HVT ventilation induced neutrophil inltration into the lungs in both genotypes, as indicated by increased lung tissue MPO activity (Fig. 6) and BALF neutrophil counts (data not shown). Lung tissue MPO activity was less in NOS3/ than in WT mice exposed to HVT ventilation. The increase in BALF neutrophils induced by HVT ventilation was similar in the two genotypes.
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The histological appearance of lungs from control WT and NOS3/ mice was not different (Fig. 7A). HVT ventilation induced inammatory cell inltration and thickening of alveolar walls of WT mice. HVT ventilation caused less inammatory cell inltration and alveolar wall thickening in NOS3/ than in WT mice. Immunohistochemistry revealed a marked increase in the number of neutrophils in the lungs of all mice exposed to HVT ventilation (Fig. 7, B and C). In NOS3/ mice exposed to HVT ventilation, lung neutrophil inltration was less than that in similarly ventilated WT mice. In control WT and NOS3/ mice, the levels of NO metabolites and nitrite/nitrate in BALF were not different (Fig. 8). HVT ventilation increased BALF NO metabolite concentrations twofold in WT mice but did not alter levels in NOS3/ mice. The levels of NOS1 and NOS2 mRNA in the lungs were not different between genotypes in control mice and were unaffected by HVT ventilation (data not shown). Effect of inhaled NO on the development of VILI in NOS3/ and WT mice. To test the hypothesis that NOS3derived NO contributes to the development of VILI, we subjected NOS3/ mice to the same HVT ventilation protocol and replenished NO in the lungs by adding a low concentration (5 ppm) of NO gas to the ventilation gas (beginning from the initiation of mechanical ventilation). We found that breathing 5 ppm NO did not increase lung injury in NOS3/ mice exposed to HVT ventilation. Lung compliance and oxygenation after 4 h of HVT ventilation were not different in NOS3/ mice ventilated with and without inhaled NO (Fig. 1B and Table 2). Total protein and IL-6 concentrations in BALF were similar in NOS3/ mice ventilated without or with 5 ppm NO, as were lung MPO activity and leukocyte accumulation (Figs. 3, 5, 7, and 8). To ascertain if a higher concentration of inhaled NO would induce injury in NOS3/ mice, we tested the effect of inhaling 50 ppm NO in mice subjected to the same HVT ventilation protocol. We found that, even at higher concentrations, inhaled NO did not increase lung injury in NOS3/ mice exposed to HVT ventilation. No differences were observed in lung compliance, oxygenation, BALF protein, or BALF IL-6 concentrations in NOS3/ mice exposed to HVT ventilation with or without 50 ppm NO (Figs. 1B, 2, 4). We examined the effect of the same concentrations of inhaled NO in WT mice subjected to HVT ventilation. Interestingly, low concentrations of inhaled NO (5 ppm) administered during HVT ventilation in WT mice ameliorated both the decrease in lung compliance and the impairment of oxygenation (Fig. 1B and Table 2). Moreover, HVT ventilation with 5 ppm inhaled NO resulted in lower levels of BALF protein and IL-6 concentrations, as well as reduced pulmonary neutrophil inltration in WT mice (Figs. 2, 4, 6, and 7). In contrast, WT mice receiving 50 ppm NO were not protected from VILI and had similar lung compliance and oxygenation as did WT mice that were not treated with NO (Fig. 1B and Table 2). BALF protein concentration was also similar in WT mice without and with inhalation of 50 ppm NO (Fig. 2). BALF IL-6 levels were higher in WT mice breathing 50 ppm NO than in those not receiving NO (Fig. 4). Together, these observations suggest that the protective effects of inhaled NO in WT mice subjected to HVT ventilation are dose dependent.
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Table 2. Arterial blood gas analysis

PaO2, mmHg D(A-a)O2, mmHg PaCO2, mmHg pH HCO 3 , mmol/l

WT-HVT NOS3/-HVT WT-HVTiNO5ppm NOS3/-HVTiNO5ppm WT-HVTiNO50ppm NOS3/-HVTiNO50ppm WT-HVTBH4/asc

184 91 297 20* 252 28# 265 34 225 20 256 33 303 24*

137 91 34 13* 76 23# 63 33 100 16 46 15 41 25*

33 8 28 3 27 6 30 10 28 1 29 4 28 10

7.33 0.07 7.36 0.05 7.38 0.05 7.38 0.08 7.39 0.08 7.35 0.04 7.42 0.05

16 3 16 2 15 2 17 3 17 1 15 2 15 5

Arterial blood gas tensions after 4 h of HVT ventilation (VT 40 ml/kg) with PEEP 1 cmH2O and FIO2 0.5 in WT and NOS3/ mice without (HVT) or with 5 ppm (HVTiNO5ppm) or 50 ppm (HVTiNO50ppm) inhaled NO, and in WT mice treated with BH4 and ascorbic acid (WT-HVTBH4/asc). PaO2 was lower and D(A-a)O2 was higher in WT-HVT mice than in NOS3/ HVT and WT-HVTBH4/asc mice (*P 0.001), and in WT-HVTiNO5ppm mice (#P 0.05); data are presented as means SD; n 5-10 per group.

Oxidative stress and sources of superoxide in WT and NOS3/ mice subjected to HVT ventilation. The observation that inhaled NO did not induce injury in NOS3/ mice, and even ameliorated VILI in WT mice at low concentrations, indicated that NOS3 does not contribute to VILI via NO production. It is known that NOS3 can become uncoupled and produce superoxide instead of NO (13) and that oxidative stress contributes to VILI (8, 29). Oxidative stress in mice subjected to HVT ventilation was evaluated by measuring the levels of lipid oxidation products, MDA and 8-isoprostane and the ratio of total to oxidized glutathione levels. HVT ventilation increased MDA levels in BALF (Fig. 9A), as well as lung and kidney tissues (Supplementary Fig. S3), in WT mice, but not in NOS3/ mice. HVT ventilation increased 8-isoprostane levels in BALF from both WT and NOS3/ mice, but levels were greater in WT mice (Fig. 9B). The ratio of total to oxidized glutathione levels was reduced in lungs of WT mice subjected to HVT ventilation but not in those from similarly treated NOS3/ mice (Fig. 9C). Together, these ndings strongly suggest that presence of NOS3 contributes to the oxidative stress caused by HVT ventilation.

To investigate whether NOS3 itself contributes to the observed increase in lung oxidative stress caused by HVT ventilation, we measured superoxide production in lung homogenates, using lucigenin-enhanced chemiluminescence, in the presence and absence of a NOS inhibitor, L-NAME. We found that superoxide production was twofold greater in lungs of WT mice subjected to HVT ventilation than in lungs from control WT mice (319 97 vs. 163 89 RLUs1mg1 protein, respectively; P 0.05). Superoxide production tended to be greater in the lungs of NOS3/ mice subjected to HVT ventilation than in the lungs of control NOS3/ mice (260 85 vs. 154 44 RLUs1mg1 protein, respectively; P 0.06). Addition of superoxide scavengers, superoxide dismutase, and Tiron, to the reaction buffer abolished the chemiluminescence signal, conrming the specicity of the assay (data not shown). L-NAME did not alter superoxide production in the lungs of control WT and NOS3/ mice or in NOS3/ mice subjected to HVT ventilation (Fig. 9B). In contrast, L-NAME markedly inhibited superoxide production in the lungs of WT mice subjected to HVT ventilation. The nding that HVT ventilation induces NOS-inhibitable superoxide production in WT lungs, but not NOS3/ lungs, strongly suggests that NOS3 itself contributes to the increased superoxide generation seen in WT mice.

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Fig. 2. Protein concentration in BALF from control WT and NOS3/ mice, and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h, without or with inhaled NO 5 ppm (iNO5ppm) or 50 ppm (iNO50ppm). BALF protein levels were greater in WT and NOS3/ mice subjected to HVT ventilation than in the corresponding controls (*P 0.001 and #P 0.05, respectively). BALF protein concentrations after HVT ventilation were less in NOS3/ mice and in WT mice breathing 5 ppm NO than in WT mice (P 0.001). N 710 per group. Boxes represent 25th-75th percentile; line and square represent median and mean, respectively; whisker represents 5th-95th percentile, and represents 1st-99th percentile. AJP-Lung Cell Mol Physiol VOL

Fig. 3. IgM concentrations in BALF from control WT and NOS3/ mice and from WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. BALF IgM levels were greater in WT and NOS3/ mice subjected to HVT ventilation than in the corresponding controls (*P 0.001 and #P 0.05, respectively). BALF IgM concentration after HVT ventilation was less in NOS3/ mice than in WT mice (P 0.001). N 710 per group. Data are presented in box plots as in Fig. 2.
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Fig. 4. IL-6 concentrations in BALF from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h, without or with inhaled NO 5 ppm (iNO5ppm) or 50 ppm (iNO50ppm). BALF IL-6 concentrations were greater in WT and NOS3/ mice subjected to HVT ventilation than in the corresponding controls (*P 0.001). BALF IL-6 concentrations after HVT ventilation were less in NOS3/ mice and in WT mice breathing 5 ppm NO than in WT mice not receiving NO (#P 0.001). BALF IL-6 concentrations after HVT ventilation were greater in WT mice breathing 50 ppm NO than in WT mice not receiving NO (P 0.01). N 3 4 per group as controls and 6 10 per group for HVT ventilation. Data are presented in box plots as in Fig. 2.

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Effect of treatment with BH4 and ascorbic acid on VILI in WT mice. Having observed that NOS3 can contribute to superoxide production by lungs of WT mice subjected to HVT ventilation, we sought to determine if BH4 and ascorbic acid, antioxidants that can prevent NOS3 uncoupling and superoxide production, would ameliorate VILI in WT mice. We found that after 4 h of HVT ventilation, lung compliance was greater in WT mice treated with BH4 and ascorbic acid than in untreated WT mice (Fig. 1B). Oxygenation was also better in BH4- and ascorbic acid-treated mice than in untreated WT mice [D(Aa)O2: 35 26 vs. 137 91 mmHg, P 0.003; Table 2]. BALF protein concentrations were lower in BH4- and ascorbic acid-treated mice subjected to HVT than in untreated, similarly ventilated WT mice (0.8 0.1 vs. 2.1 1.0 mg/ml, P 0.002), as were BALF IL-6 levels (0.7 0.3 vs. 1.0 0.1 ng/ml, P 0.03).
DISCUSSION

The main nding of this study is that NOS3 is an important participant in the pathogenesis of murine VILI in vivo. HVT induced a greater decrease in lung compliance and a more marked increase in lung permeability, inammation, and oxidative stress in WT than in NOS3/ mice. Replenishment of NO by adding NO to the inhaled gas did not increase lung injury in NOS3/ mice exposed to HVT ventilation. HVT ventilation induced L-NAME-inhibitable superoxide production in WT but not in NOS3/ mice. Finally, VILI was reduced in WT mice by pretreatment with the antioxidants BH4 and ascorbic acid that can prevent NOS3 uncoupling. Together, our data support the hypothesis that NOS3 contributes to VILI in a NO-independent manner, by increasing the production of superoxide likely by uncoupled NOS3.
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Fig. 5. MIP-2 (A) and TNF (B) concentrations in BALF, and IL-6 concentrations in plasma (C) from WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. In control mice of both genotypes, MIP-2 and TNF were not detected in BALF, and IL-6 was not detected in plasma. After HVT ventilation, BALF MIP-2 and TNF concentrations were increased in both genotypes, but less in NOS3/ mice than in WT mice (*P 0.01). Plasma IL-6 concentrations after HVT ventilation were less in NOS3/ mice than in WT mice (*P 0.001). N 3 4 per group as controls, and 6 10 per group for HVT ventilation. Data are presented in box plots as in Fig. 2.

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Fig. 6. Lung myeloperoxidase (MPO) activity from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h, with or without 5 ppm inhaled NO (iNO5ppm). Lung MPO activity was greater in WT and NOS3/ mice subjected to HVT ventilation than in the corresponding controls (*P 0.001 and #P 0.01, respectively). Lung MPO activity after HVT ventilation was less in NOS3/ mice and in WT mice breathing 5 ppm NO than in WT mice not receiving NO (P 0.01 and P 0.001, respectively). Data are expressed as MPO activity units per milligram of tissue; n 3 4 per group for control and 6 9 per group for HVT ventilation. Data are presented in box plots as in Fig. 2.

In the present study, we used a mouse model in which HVT ventilation (40 ml/kg) consistently disrupted the alveolar-capillary barrier (resulting in high-permeability pulmonary edema) and induced inammation, hallmarks of VILI (14, 20, 29, 42). We observed that HVT ventilation-induced pulmonary edema was greater in WT mice than in NOS3/ mice, as indicated by the downward shift of the pressure-volume curve and impaired arterial oxygenation. Total protein and IgM concentrations in BALF were higher in WT than in NOS3/ mice after HVT ventilation, consistent with more marked disruption of the alveolar-capillary barrier in WT mice. Moreover, the levels of proinammatory cytokines in BALF and lung neutrophil inltration were greater in WT that in NOS3/ mice exposed to HVT ventilation. Our in vivo results demonstrate that NOS3 contributes to both HVT-induced pulmonary edema and lung inammation. HVT ventilation is associated with increased levels of NO metabolites in BALF (15, 17). In our study, BALF concentrations of NO metabolites were increased in WT but not in NOS3/ mice subjected to HVT ventilation. Pulmonary NOS1 and NOS2 mRNA levels were not different in WT and NOS3/ mice suggesting that the observed differences in BALF NO metabolite concentrations between the two genotypes were attributable to NOS3. To determine whether reduced pulmonary NO levels were responsible for the protective effects of NOS3 deciency, we examined the impact of replenishing pulmonary NO levels in NOS3/ mice subjected to HVT ventilation. We studied the impact of a low dose of inhaled NO (5 ppm), similar to the concentration previously used to restore the WT phenotype in NOS3/ mice (5), and a higher dose (50 ppm), similar to that previously used to restore the WT phenotype in NOS2/ mice (39, 41). We found that addition of inhaled NO at either low or high concentrations had no effect on the response of NOS3/
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mice to HVT ventilation. These ndings suggest that NO produced by NOS3 is insufcient to account for the greater VILI seen in WT mice than in NOS3/ mice. We also examined the effect of breathing the same concentrations of NO in WT mice subjected to HVT ventilation. Interestingly, we found that breathing 5 ppm NO protected WT mice from the development of VILI. Our results are in agreement with previous studies showing that low concentrations of inhaled NO were protective in other models of lung injury (6, 18, 21, 32, 35). In contrast, we found that breathing higher concentrations of NO did not prevent lung injury in WT mice subjected the HVT ventilation. These results suggest that high lung NO levels have deleterious effects that can overwhelm the positive effects of low NO concentrations. The contribution of NOS3 in VILI has been examined in other studies with conicting results. Consistent with our study, Schmidt et al. (36), using isolated-perfused mouse lungs, found that NOS3 deciency reduces HVT-induced pulmonary edema and suggested that NO and cyclic GMP signaling contribute to HVT-induced barrier disruption. In contrast, Peng et al. (31) recently reported that pulmonary edema was more marked in NOS3/ than in WT mice. Takenaka et al. (38) observed that transgenic overexpression of NOS3, leading to increased pulmonary NO levels, protected mice from VILI. We found that increasing pulmonary NO levels with low concentrations of inhaled NO protected WT mice from VILI. It is of note that Peng and colleagues and Takenaka and colleagues both used a VT of 20 ml/kg, whereas we used a VT of 40 ml/kg. In our pilot studies, we found that ventilation with 40 ml/kg caused a greater and more consistent degree of lung injury and inammation than did 20 ml/kg. In our model, the deleterious effects of NOS3 in VILI did not appear to be mediated by NO, leading us to investigate whether another NOS3 product could contribute to HVTinduced lung injury. It is known that NOS can produce superoxide instead of NO when the levels of BH4 or L-arginine are insufcient (13), a process referred to as NOS uncoupling. Superoxide derived from uncoupled NOS3 contributes to vascular dysfunction both in animal models and in human beings with hypertension, hypercholesterolemia, and diabetes mellitus (13). HVT ventilation could induce NOS3 uncoupling by depleting BH4 and/or arginine. HVT ventilation may deplete arginine by inducing expression of arginases (2), but in our model, HVT did not increase lung arginase I and II gene expression (data not shown). Alternatively, ROS generated in the lungs during HVT ventilation by xanthine oxidase and neutrophil NADPH oxidase (1, 10, 33) may inactivate BH4 (22), thereby uncoupling NOS3 (23) and leading to additional superoxide production. In our study, HVT ventilation increased oxidative stress in WT mice, as indicated by increased MDA and 8-isoprostane levels in BALF, reduced total to oxidized glutathione levels in lung tissue, and increased MDA in both lungs and kidneys. NOS-inhibitable superoxide production in lungs from WT but not from NOS3/ mice exposed to HVT ventilation suggests that NOS3 itself can contribute to lung superoxide production. Superoxide, generated by NOS and other enzymes, likely contributes to the development of VILI via several mechanisms. Superoxide can induce lipid peroxidation leading to cell membrane destruction (37) and increased vascular permeability (40). Superoxide can increase ROS production from neutrophils and macrophages and affect the
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Fig. 7. Histology and immunohistochemistry for neutrophils in lung sections from control WT and NOS3/ mice and from WT and NOS3/ mice subjected to 4 h of HVT ventilation (40 ml/kg) with or without 5 ppm (iNO5ppm) inhaled NO. A: representative photographs of lung sections stained with hematoxylin and eosin and presented in 40 original magnication. Control WT (1) and NOS3/ (2) mice had preserved lung parenchymal architecture, and there were no differences observed between genotypes. HVT ventilation resulted in increased cellular inltration and alveolar wall thickening in WT mice (3) that was less marked in NOS3/ mice (4) and in WT mice breathing 5 ppm NO (5). Breathing 5 ppm NO did not alter the histological appearance of lungs from NOS3/ mice exposed to HVT ventilation (6). B: representative photographs of lung sections reacted with anti-mouse neutrophil monoclonal antibody are presented in 40 original magnication. Few neutrophils (arrows) were observed in lungs from control WT (7) and NOS3/ (8) mice. HVT ventilation induced neutrophil inltration to a greater degree in WT (9) than in NOS3/ (10) mice. Breathing 5 ppm NO reduced neutrophil accumulation in WT mice subjected to HVT ventilation (11). Neutrophil inltration after HVT ventilation was similar in NOS3/ with (12) and without inhaled NO. White arrow shows neutrophils inside small vessels. C: quantication of neutrophil immunostaining in lungs from WT and NOS3/ mice. Neutrophil inltration was greater in WT and NOS3/ mice subjected to HVT ventilation than in the corresponding controls (*P 0.001); neutrophil inltration after HVT ventilation was less in NOS3/ mice and in WT mice breathing 5 ppm NO than in WT mice not receiving NO (#P 0.01 for both). Data represent means SD of number of neutrophils per high-power magnication eld counted in at least 50 elds (40) for each group.

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activity of other ROS-producing enzymes (11, 28). By this means, NOS3 uncoupling can contribute to VILI by amplifying ROS production from other enzymes, such as NADPH oxidase and xanthine oxidase, and increase oxidative stress. We found that treatment with BH4 and ascorbic acid, antioxidants that can prevent NOS3 uncoupling (22, 27), ameliorated VILI in WT mice. BH4 serves as an electron donor in NO synthesis and is indispensable for NOS3 function (1), whereas ascorbic acid recycles the oxidized BH3 radical to BH4 (22, 27). Although BH4 and ascorbic acid may protect against VILI via mechanisms other than NOS3 coupling, our ndings support the hypothesis that NOS3-derived superoxide plays an important role in VILI. Our study has several limitations. To evaluate the role of NOS3-derived NO in VILI, we replenished lung NO levels by adding NO gas to the gas mixture used to ventilate the mice we studied. Although the location and concentration of NO may differ in WT mice expressing NOS3 and in NOS3/ mice
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breathing NO, we and others have successfully used inhaled NO to restore a WT phenotype in NOS-decient mice (5, 39, 41). Moreover, use of inhaled NO permitted us to discriminate between the roles of NO and superoxide in vivo in a manner that was not achievable with NOS inhibitors, which block the synthesis of both NO and superoxide by NOS, or with NOdonor compounds, which can cause systemic hypotension. We found that NOS3 contributes to oxidative stress and superoxide production in lungs of WT mice subjected to HVT ventilation, which we attributed to NOS3 uncoupling. We did not nd increased monomerization of NOS3 (data not shown), which has been associated with NOS3 uncoupling (27). However, other investigators have reported that puried NOS monomers are inactive (4, 7) and that the presence of NOS monomers is not required for NOS uncoupling (13). In contrast to the observations of Peng and colleagues (31), we also did not nd increased levels of nitrotyrosine (data not shown), an index of peroxynitrite generation, in the lungs of mice ventilated with
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Fig. 8. BALF concentrations of NO metabolites (nitrite/nitrate, NOx) from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. BALF NOx levels were greater in WT mice subjected to HVT ventilation than in WT controls (*P 0.001). BALF NOx levels after HVT ventilation were less in NOS3/ mice than in WT mice (#P 0.01). N 5 8 per group. Data are presented in box plots as in Fig. 2.

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high tidal volumes. These differing results may be attributable to differences in the mouse models of VILI and/or the methods used to measure lung nitrotyrosine levels. The observation that HVT ventilation is associated with increased superoxide production by NOS3 provides a novel therapeutic target for VILI. Pharmacological interventions aimed at preventing NOS3 uncoupling, such as administration of BH4 or 5-methyltetrahydrofolate (3, 27), that have shown promising results of experimental studies in vascular disease, may also play a role in the prevention and treatment of VILI. In summary, our data suggest that NOS3 contributes to the alveolar-capillary barrier dysfunction, pulmonary inamma-

Fig. 9. A: BALF concentrations of malondialdehyde (MDA) from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. BALF MDA levels were greater in WT mice subjected to HVT ventilation than in WT controls (*P 0.01), but were unchanged in NOS3/ mice. BALF MDA levels after HVT ventilation were less in NOS3/ mice than in WT mice (#P 0.01). Data are presented in box plots as in Fig. 2; n 6 8 per group. B: BALF concentrations of 8-isoprostane from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. BALF 8-isoprostane levels were greater in WT and NOS3/ mice subjected to HVT ventilation than in corresponding controls (*P 0.001 and #P 0.01, respectively). BALF MDA levels after HVT ventilation were less in NOS3/ than WT mice (P 0.01). Data are presented in box plots as in Fig. 2; n 5 per group. C: the ratio of total to oxidized glutathione levels (GSH/GSSG) in lung tissues from control WT and NOS3/ mice, and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. GSH/GSSG was less in WT mice subjected to HVT ventilation than in WT controls (*P 0.05). HVT ventilation did not reduce GSH/GSSG in NOS3/ mice, and GSH/ GSSG was greater in NOS3/ mice subjected to HVT ventilation than in similarly ventilated WT mice (*P 0.05). Data are presented in box plots as in Fig. 2; n 5 per group. D: NOS-derived superoxide production in lung homogenates from control WT and NOS3/ mice and WT and NOS3/ mice ventilated with HVT (40 ml/kg) for 4 h. NOS-derived superoxide production was estimated from the difference in lucigenin-enhanced chemiluminescence (LEC) measured in the absence and presence of L-NAME (1 mM). L-NAME-inhibitable superoxide production was greater in WT mice subjected to HVT ventilation than in WT control mice and in similarly ventilated NOS3/ mice (*P 0.01 for both). Data are expressed as relative light units (RLU) per second per milligram of protein, n 5 per group. Data are presented in box plots as in Fig. 2. AJP-Lung Cell Mol Physiol VOL
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NOS3 CONTRIBUTES TO VILI 13. Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113: 1708 1714, 2006. 14. Frank JA, Matthay MA. Science review: mechanisms of ventilatorinduced injury. Crit Care 7: 233241, 2003. 15. Frank JA, Pittet JF, Lee H, Godzich M, Matthay MA. High tidal volume ventilation induces NOS2 and impairs cAMP-dependent air space uid clearance. Am J Physiol Lung Cell Mol Physiol 284: L791L798, 2003. 16. Haddad JJ. Redox regulation of pro-inammatory cytokines and IkappaB-alpha/NF-kappaB nuclear translocation and activation. Biochem Biophys Res Commun 296: 847856, 2002. 17. Hammerschmidt S, Schiller J, Kuhn H, Meybaum M, Gessner C, Sandvoss T, Arnold K, Wirtz H. Inuence of tidal volume on pulmonary NO release, tissue lipid peroxidation and surfactant phospholipids. Biochim Biophys Acta 1639: 1726, 2003. 18. Hataishi R, Kobayashi H, Takahashi Y, Hirano S, Zapol WM, Jones RC. Myeloperoxidase-associated tyrosine nitration after intratracheal administration of lipopolysaccharide in rats. Anesthesiology 97: 887895, 2002. 19. Holter JF, Weiland JE, Pacht ER, Gadek JE, Davis WB. Protein permeability in the adult respiratory distress syndrome. Loss of size selectivity of the alveolar epithelium. J Clin Invest 78: 15131522, 1986. 20. Hong SB, Huang Y, Moreno-Vinasco L, Sammani S, Moitra J, Barnard JW, Ma SF, Mirzapoiazova T, Evenoski C, Reeves RR, Chiang ET, Lang GD, Husain AN, Dudek SM, Jacobson JR, Ye SQ, Lussier YA, Garcia JG. Essential role of pre-B-cell colony enhancing factor in ventilator-induced lung injury. Am J Respir Crit Care Med 178: 605617, 2008. 21. Kang JL, Park W, Pack IS, Lee HS, Kim MJ, Lim CM, Koh Y. Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-B and inammation. J Appl Physiol 92: 795801, 2002. 22. Kuzkaya N, Weissmann N, Harrison DG, Dikalov S. Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric-oxide synthase. J Biol Chem 278: 22546 22554, 2003. 23. Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG. Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111: 12011209, 2003. 24. Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG. Endothelial regulation of vasomotion in apoE-decient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 103: 12821288, 2001. 25. Liu R, Hotta Y, Graveline AR, Evgenov OV, Buys ES, Bloch KD, Ichinose F, Zapol WM. Congenital NOS2 deciency prevents impairment of hypoxic pulmonary vasoconstriction in murine ventilator-induced lung injury. Am J Physiol Lung Cell Mol Physiol 293: L1300 L1305, 2007. 26. Moens AL, Kass DA. Tetrahydrobiopterin and cardiovascular disease. Arterioscler Thromb Vasc Biol 26: 2439 2444, 2006. 27. Moens AL, Takimoto E, Tocchetti CG, Chakir K, Bedja D, Cormaci G, Ketner EA, Majmudar M, Gabrielson K, Halushka MK, Mitchell JB, Biswal S, Channon KM, Wolin MS, Alp NJ, Paolocci N, Champion HC, Kass DA. Reversal of cardiac hypertrophy and brosis from pressure overload by tetrahydrobiopterin: efcacy of recoupling nitric oxide synthase as a therapeutic strategy. Circulation 117: 2626 2636, 2008. 28. Muzaffar S, Shukla N, Angelini GD, Jeremy JY. Superoxide autoaugments superoxide formation and upregulates gp91(phox) expression in porcine pulmonary artery endothelial cells: inhibition by iloprost. Eur J Pharmacol 538: 108 114, 2006. 29. Oeckler RA, Hubmayr RD. Ventilator-associated lung injury: a search for better therapeutic targets. Eur Respir J 30: 1216 1226, 2007. 30. Peng X, Abdulnour RE, Sammani S, Ma SF, Han EJ, Hasan EJ, Tuder R, Garcia JG, Hassoun PM. Inducible nitric oxide synthase contributes to ventilator-induced lung injury. Am J Respir Crit Care Med 172: 470 479, 2005. 31. Peng XQ, Damarla M, Skirball J, Nonas S, Wang XY, Han EJ, Hasan EJ, Cao X, Boueiz A, Damico R, Tuder RM, Sciuto AM, Anderson DR, Garcia JG, Kass DA, Hassoun PM, Zhang JT. Protective role of PI3-kinase/Akt/eNOS signaling in mechanical stress through inhibition of p38 mitogen-activated protein kinase in mouse lung. Acta Pharmacol Sin 31: 175183, 2010.
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tion, and oxidative stress associated with VILI. NOS3-derived NO appears insufcient to cause VILI, as inhaled NO does not worsen VILI in NOS3/ mice, and, in fact, at low concentrations, NO can ameliorate VILI in WT mice. These ndings suggest that NOS3, and in particular superoxide generation by NOS3, may represent a novel therapeutic target for the prevention of VILI.
ACKNOWLEDGMENTS We thank Drs. Yasuko Nagasaka, Patricio Leyton, and Emmanuel Buys and Michael J. Raher, Kristen Rauwerdink, Yuko Beppu, and Dr. Rong Liu for advice and skillful assistance; Dr. Rosemary Jones for advice interpreting lung histology; and Dr. Hui Zheng for assistance in statistical analysis. GRANTS This work was supported, in part, by National Heart, Lung, and Blood Institute Grant HL-42397 (to W. M. Zapol) and by a sponsored research agreement between the Massachusetts General Hospital (MGH) and IKARIA. DISCLOSURES The MGH has obtained patents relating to the use of inhaled nitric oxide and has licensed them to IKARIA and Linde Gas Therapeutics, Lidingo, Sweden. W. M. Zapol receives royalties and K. D. Bloch has received grants from IKARIA. REFERENCES 1. Abdulnour RE, Peng X, Finigan JH, Han EJ, Hasan EJ, Birukov KG, Reddy SP, Watkins JE 3rd, Kayyali US, Garcia JG, Tuder RM, Hassoun PM. Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways. Am J Physiol Lung Cell Mol Physiol 291: L345L353, 2006. 2. Altemeier WA, Matute-Bello G, Gharib SA, Glenny RW, Martin TR, Liles WC. Modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation. J Immunol 175: 3369 3376, 2005. 3. Antoniades C, Shirodaria C, Warrick N, Cai S, de Bono J, Lee J, Leeson P, Neubauer S, Ratnatunga C, Pillai R, Refsum H, Channon KM. 5-Methyltetrahydrofolate rapidly improves endothelial function and decreases superoxide production in human vessels: effects on vascular tetrahydrobiopterin availability and endothelial nitric oxide synthase coupling. Circulation 114: 11931201, 2006. 4. Baek KJ, Thiel BA, Lucas S, Stuehr DJ. Macrophage nitric oxide synthase subunits. Purication, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J Biol Chem 268: 21120 21129, 1993. 5. Balasubramaniam V, Maxey AM, Morgan DB, Markham NE, Abman SH. Inhaled NO restores lung structure in eNOS-decient mice recovering from neonatal hypoxia. Am J Physiol Lung Cell Mol Physiol 291: L119 L127, 2006. 6. Barbotin-Larrieu F, Mazmanian M, Baudet B, Detruit H, Chapelier A, Libert JM, Dartevelle P, Herve P. Prevention of ischemia-reperfusion lung injury by inhaled nitric oxide in neonatal piglets. J Appl Physiol 80: 782788, 1996. 7. Bauersachs J, Schafer A. Tetrahydrobiopterin and eNOS dimer/monomer ratioa clue to eNOS uncoupling in diabetes? Cardiovasc Res 65: 768 769, 2005. 8. Birukov KG. Cyclic stretch, reactive oxygen species, and vascular remodeling. Antioxid Redox Signal 11: 16511667, 2009. 9. Boueiz A, Hassoun PM. Regulation of endothelial barrier function by reactive oxygen and nitrogen species. Microvasc Res 77: 26 34, 2008. 10. DeCoursey TE, Morgan D, Cherny VV. The voltage dependence of NADPH oxidase reveals why phagocytes need proton channels. Nature 422: 531534, 2003. 11. Forman HJ, Torres M. Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am J Respir Crit Care Med 166: S4 S8, 2002. 12. Forstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H. Nitric oxide synthase isozymes. Characterization, purication, molecular cloning, and functions. Hypertension 23: 11211131, 1994. AJP-Lung Cell Mol Physiol VOL

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