|
|
Am J Respir Crit Care Med. 2005 August 15; 172(4): 470–479. Published online 2005 June 3. doi: 10.1164/rccm.200411-1547OC. | PMCID: PMC2718528 |
Inducible Nitric Oxide Synthase Contributes to Ventilator-induced Lung Injury Xinqi Peng,* Raja-Elie E. Abdulnour,* Saad Sammani, Shwu-Fan Ma, Eugenia J. Han, Emile J. Hasan, Rubin Tuder, Joe G. N. Garcia, and Paul M. Hassoun Division of Pulmonary and Critical Care Medicine, Department of Pathology, and the Center for Translational Respiratory Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland Received November 19, 2004; Accepted May 20, 2005. Rationale: Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation. Objectives: This study investigated changes in lung NOS expression and activity in a mouse model of ventilator-induced lung injury. Methods: C57BL/6J (wild-type [WT]) and iNOS-deficient (iNOS−/−) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay. Results: iNOS mRNA and protein expression was absent in iNOS−/− mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS−/− mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS−/− mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS−/− mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS−/− mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine. Conclusion: Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury. Keywords: inducible nitric oxide synthase, lung permeability, mechanical ventilation Nitric oxide (NO) is involved in many physiologic and pathologic conditions, such as blood vessel relaxation, neurotransmission, and host defense. NO also plays a critical role in tissue injury in the context of various inflammatory conditions ( 1). NO is produced by three isoforms (neuronal, endothelial, and inducible) of NO synthase (NOS). Endothelial NOS (eNOS), which is calcium/calmodulin (Ca 2+/CaM)-dependent and activated by agonists (e.g., acetylcholine), produces a low level of NO output, whereas inducible NOS (iNOS) is independent of Ca 2+/CaM, is transcriptionally regulated by proinflammatory products (e.g., LPS) and cytokines (interleukin 1β [IL-1β], tumor necrosis factor α [TNF-α], IFN-γ), and results in sustained and elevated release of NO ( 2). Therefore, overproduction of NO, in particular in the setting of superoxide production ( 3), leads to oxidative stress and tissue injury in conditions such as endotoxin-induced acute lung injury (ALI) ( 4). ALI is characterized by a severe inflammatory process, profound hypoxia, and respiratory failure most often requiring mechanical ventilation (MV) for life support. However, it is now recognized that mechanical stress related to positive-pressure MV may cause or aggravate ALI, in particular when high V t (HV t) ventilation is applied ( 5). Furthermore, a systemic inflammatory response may be elicited by MV during recruitment or derecruitment of collapsed lung units and when alveolar regions are overdistended ( 6). Several investigators, including our group, have demonstrated increased activity of certain signaling pathways in animal models of MV ( 7– 10). A recent study by Frank and colleagues ( 11) demonstrated increased lung iNOS protein expression and total nitrite levels in bronchoalveolar lavage (BAL) fluid, and a decrease in airspace fluid clearance in rats ventilated with HV t as compared with low V t (LV t). Inhibition of iNOS prevented the decrease in airspace fluid clearance, suggesting a role for reactive nitrogen species in the pathogenesis of ventilator-associated lung injury (VILI). In contrast, work by Choi and colleagues ( 12) demonstrated an increase in eNOS protein expression in lungs and kidneys in a rat model of HV t (20 ml/kg for 2 hours) but without detectable iNOS expression in this model. Although these investigators did not measure specific NOS activity in their model, they demonstrated that the nonspecific NOS inhibitor N-nitro- l-arginine methyl ester attenuated lung and kidney microvascular leakage ( 12). The aim of this study was to investigate changes in lung NOS expression and activity in a mouse model of VILI. We also assessed the specific role of iNOS in the development of ventilator-induced capillary leakage with the use of iNOS-deficient mice and specific chemical inhibition. Our results indicate that iNOS expression and activity are significantly upregulated by HV t ventilation, are accompanied by nitrotyrosine deposition, mainly in capillary endothelial cells, and correlate with increased lung capillary permeability. Furthermore, iNOS-deficient mice were protected from pulmonary edema in response to MV with HV t. Finally, specific chemical inhibition of iNOS activity with aminoguanidine (AG) abrogated the capillary leakage produced by MV in wild-type animals. Some of the results of these studies have been previously published in abstract form ( 13). Johns Hopkins University Institutional Animal Care and Use Committee approved all animal protocols. Additional method details are reported in the online supplement. Animal Preparation Male C57BL/6 and iNOS knock-out mice, 8 to 10 weeks old, were anesthetized and underwent tracheotomy, and the jugular vein was cannulated. The mice were then exposed to LVt (7 ml/kg, 120 breaths/minute) and HVt (20 ml/kg, 60 breaths/minute) MV for 2 hours. In some experiments, animals were pretreated with AG (15 mg/kg, intraperitoneally), a selective iNOS inhibitor (Sigma, St. Louis, MO), 1 hour before exposure to MV. The adequacy of MV settings on gas exchange was confirmed in preliminary experiments by arterial blood gases analysis (Instrumentation Laboratories, Lexington, MA), which revealed stable levels of arterial oxygen and carbon dioxide. Immunoblotting Western blotting was performed as previously described ( 14, 15) after tissue homogenates were prepared and protein quantified by bicinchoninic acid assay (Pierce, Rockford, IL). Assessment of Lung Capillary Leakage Evans blue dye albumin (EBA; 20 mg/kg) was injected into the internal jugular vein 30 minutes before the termination of the experiment to assess vascular leak, as previously described ( 10, 16, 17). Determination of BAL Protein, Total Cell Counts, and Inflammatory Cytokines BAL was performed by intratracheal injection of 1 ml of Hank's balanced salt solution followed by gentle aspiration. The recovered fluid was processed for protein and cell count, as described previously ( 16), and measurement of cytokines was performed by specific ELISA. Lung Immunohistochemistry After removal of paraffin, lung tissue sections were incubated for 1 hour at room temperature with diluted monoclonal antinitrotyrosine (Transduction Laboratories, Lexington, KY) or antineutrophil (Dako Corporation, Carpinteria, CA) primary antibodies. The immunohistochemical reaction was visualized by incubation with 0.05% diaminobenzidine (DAB). Lung Immunofluorescence Tissue sections were incubated with anti-iNOS, antinitrotyrosine, anti–surfactant protein C, and anti-CD34 primary antibodies. The samples were then incubated with fluorescein isothiocyanate–labeled secondary antibodies and visualized under fluorescent microscopy. Lung Morphology Lungs from animals in each experimental group were inflated under a pressure of 25 cm H 2O with 0.5% of low melting agarose (Invitrogen, Carlsbad, CA) for histologic evaluation by hematoxylin and eosin staining as previously described ( 18, 19). Measurement of NOS Activity NOS activity was determined by measuring the conversion of [ 3H] l-arginine to [ 3H] l-citrulline using a modification of the Bredt and Snyder procedure ( 20). Calcium-dependent eNOS activity was determined by the addition of 0.6 mM CaCl 2, whereas the addition of 1 mM ethyleneglycol- bis-(β-aminoethyl ether)- N, N′-tetraacetic acid allowed the determination of the calcium-independent iNOS activity. Enzymatic activity is reported as pmol l-citruline/mg protein/minute. Semiquantitative Reverse Transcription–Polymerase Chain Reaction Total RNA was isolated and then used for a one-step semiquantitative reverse transcription–polymerase chain reaction (Invitrogen). Primers specific for mouse iNOS were designed on the basis of published cDNA sequences ( 22). Primers for 18S ribosomal RNA were used as internal positive controls under the same conditions. The steady-state mRNA levels are expressed in arbitrary units as the ratio of iNOS/18S expression ( 23). Statistical Analysis All experiments were repeated at least three times. Representative experiments are shown with values expressed as means ± SE, with n ![[gt-or-equal, slanted]](/corehtml/pmc/pmcents/ges.gif) 3 for each condition. Data were analyzed by two-way analysis of variance with Bonferroni correction, and significance in all cases was defined at p < 0.05. Effect of MV on Peak Airway Pressure We have previously demonstrated that MV with HV t (17 ml/kg) for 2 hours causes significant pulmonary vascular leakage in C57/BL6 mice, an effect that was attenuated by intravenous treatment with the lipid growth factor sphingosine 1-phosphate ( 10). We used a similar animal model of VILI to examine the effects of MV on NOS gene expression and activity. C57BL/6J and iNOS-deficient (iNOS −/−) mice obtained from the same strain background were exposed to MV for 2 hours at LV t (7 ml/kg) or HV t (20 ml/kg). Peak airway pressure was monitored continuously as detailed in M ethods. As shown in , peak airway pressures were significantly higher in wild-type and iNOS −/− mice exposed to HV t as compared with LV t. However, in both LV t and HV t groups, there was a small but significant increase in airway pressure (average of 1 and 3 cm H 2O for LV t and HV t, respectively) over time (at 60 and 120 minutes) compared with baseline (Time 0) value. There were no trend differences in peak airway pressures between wild-type and iNOS −/− mice (). Effect of MV with HVT on iNOS Gene Expression In a separate group of animals, total lung mRNA and protein were collected for semiquantitative polymerase chain reaction and Western blot analysis after exposure to MV. As expected, there was no iNOS mRNA expression in iNOS−/− mice breathing spontaneously or exposed to MV (results not shown). As shown in , iNOS mRNA expression in spontaneously breathing (sham-treated) C57BL/6J control mice was minimal but increased significantly (~ 2.7-fold) in animals exposed to HVt. iNOS protein expression was undetectable in iNOS−/− mice breathing spontaneously or exposed to HVt and minimal in C57BL/6J mice breathing spontaneously (). However, although LVt exposure caused a nonsignificant increase in iNOS protein, HVt exposure produced a 3.5-fold increase (p < 0.05 vs. sham) in this expression in C57BL/6J mice (). Changes in iNOS protein produced over time were examined in a separate group of C57BL/6J mice exposed to HVt MV and were time-dependent (). Using immunohistochemistry with antibodies specific for iNOS and endothelial (anti-CD34) and epithelial (anti–surfactant protein C) cells, iNOS immunostaining was prominent in C57BL/6J mice exposed to HVt () and was localized predominantly in pulmonary vessels () and endothelial cells (), and less markedly in epithelial cells (). Effect of MV on eNOS Protein Expression The effect of HVt MV on eNOS expression was also assessed in C57BL/6J and iNOS−/− mice. As expected, eNOS protein was constitutively expressed in spontaneously breathing (sham-treated) C57BL/6J and iNOS−/− mice (). In comparison, this expression was significantly decreased in C57BL/6J mice, but relatively unchanged in iNOS−/− mice, in response to HVt (). Increased iNOS Activity by MV We also examined lung eNOS and iNOS activities in C57BL/6J and iNOS−/− mice exposed to HVt MV. As shown in , there was a significant increase in lung iNOS activity in C57BL/6J mice in response to HVt as compared with spontaneously breathing control animals. As expected, there was no significant Ca2+-independent lung NOS activity in iNOS−/− mice () and no change in response to HVt. However, eNOS activity tended to decrease in C57BL/6J mice, but increased significantly in iNOS−/− mice in response to HVt, as compared with control counterparts (). Deficiency of iNOS Protects against Ventilator-induced VILI We assessed lung injury (by histology and BAL protein and cell count) and pulmonary vascular leakage (by the EBA technique) in a separate group of C57BL/6J and iNOS−/− mice exposed to MV. There were no striking histologic changes in lungs of C57BL/6J mice exposed to LVt as compared with control animals (results not shown). However, HVt caused ALI as suggested by parenchymal cell infiltration as well as hemorrhage in C57BL/6J mice, findings that were minimal in iNOS−/− mice (). In addition, there was a significant increase in BAL total cell count in C57BL/6J mice in response to HVt (50 ± 5 vs. 36.4 ± 4 cells/ml of BAL fluid in HVt compared with the sham group, respectively; p < 0.05), but no change in iNOS−/− mice (28.7 ± 3 vs. 30.4 ± 4 cells/ml in HVt compared with the sham group, respectively). Similarly, the number of neutrophils present in the lung parenchyma was significantly increased in C57BL/6J mice, but unchanged in iNOS−/− mice, in response to HVt exposure (). Furthermore, there was accumulation of nitrotyrosine product (as assessed by immunohistochemistry [] and digital counting of the immunostaining images []) in the lungs of C57BL/6J, but not in iNOS−/− mice, in response to HVt. As shown in , accumulation of nitrotyrosine was predominant in endothelial and epithelial cells, which were sites of increased iNOS expression (). However, there was no difference in BAL protein in C57BL/6J and iNOS−/− mice in response to HVt (). Finally, EBA accumulation in the lungs of C57BL/6J mice exposed to LVt was trivial, but increased significantly in response to HVt. In sharp contrast, HVt caused no significant EBA leakage in iNOS−/− mice (). To confirm the pathogenic role of iNOS in capillary permeability in this model, C57BL/6J and iNOS−/− mice were exposed to MV with HVt with or without pretreatment with AG, a specific chemical inhibitor of iNOS. AG treatment had no effect on EBA lung accumulation in control, nonventilated, wild-type or iNOS-deficient mice (results not shown); however, it completely abrogated increased EBA lung accumulation in wild-type C57BL/6J mice in response to MV without affecting EBA accumulation in iNOS−/− mice. The latter mice were again protected from increased capillary permeability (), as previously demonstrated. The consequences of NO release after induction of iNOS, an enzyme generally expressed under pathologic conditions, have been implicated in the pathogenesis of several diseases, including asthma, ALI, and circulatory shock. Although iNOS is generally believed to be induced by cytokines and inflammatory products, there have been recent reports of regulation of this enzyme by mechanical forces. For example, iNOS expression is increased in systemic smooth muscle cells exposed to shear stress ( 24) and in osteoblasts in response to mechanical loading ( 25). Similarly, eNOS expression is increased in bovine aortic endothelial cells in response to oscillatory shear stress ( 26) and in pulmonary vascular cells in response to circumferential stretch ( 27). On the basis of these findings, we determined whether cyclic mechanical stress induced by MV could alter the expression of lung iNOS and eNOS, and whether iNOS expression contributed to the development of capillary permeability related to VILI. Our study indicates that iNOS mRNA and protein expression is significantly increased with 2 hours of MV, correlates with the degree of alveolar distension (V t), and is accompanied by a significant increase in iNOS activity in C57BL/6J mice. eNOS protein expression was decreased in wild-type animals and relatively unchanged in iNOS −/− mice. However, there was a significant increase in eNOS activity in iNOS −/− mice in response to HV t. We speculate that this upregulation may be related to a potential direct effect of mechanical stress on eNOS expression, the lack of iNOS-derived NO, and/or the relative absence of nitrosative stress in the iNOS −/− mice. Indeed, changes in cell redox status (e.g., oxidative or nitrosative stress) have been reported as a mechanism of downregulation of eNOS activity through denitrosylation of eNOS ( 28) and therefore may explain a lack of similar increase in eNOS activity in the C57BL/6J mice in response to HV t. MV with a V t of 20 ml/kg produced ALI in wild-type animals as assessed by histologic changes, increased total cell count in BAL, parenchymal infiltration of neutrophils, evidence of nitrosative stress (accumulation of nitrotyrosine product), and increased capillary leakage. However, there was no evidence of epithelial injury (as reflected by a lack of increase in BAL protein), which is not surprising considering that efflux of protein into the alveolar space is significantly delayed (i.e., several hours) in relationship to increased capillary permeability, as demonstrated by Quinn and coworkers ( 29) in a rat model of MV using V ts similar to those used in the present study. There was overall less pulmonary injury (as reflected by a more modest increase in BAL cells and extravasation of EBA dye) in the present model as compared with a model of endotoxin inhalation ( 16). However, all changes were significantly attenuated in iNOS −/− mice, and pulmonary capillary leakage was prevented in these mice and in wild-type mice pretreated with the iNOS inhibitor AG, strongly suggesting that the increase in iNOS expression in C57BL/6J mice contributes to lung inflammation, nitrosative stress, and capillary leakage in this model. The role of NOS in VILI has been the subject of previous investigations. A recent report by Frank and colleagues ( 11) demonstrated an increase in lung iNOS protein expression and BAL nitrite and nitrate levels in rats ventilated with HV t (30 ml/kg) for 1 hour followed by LV t (7 ml/kg) for 2 hours. Furthermore, inhibition of iNOS activity prevented the decrease in airspace fluid clearance caused by MV. In contrast, Choi and colleagues ( 12) demonstrated an increase in eNOS protein expression in lungs and kidneys in a rat model of HV t (20 ml/kg for 2 hours), however, with no detectable iNOS expression. Treatment of the animals with L-NAME, a nonspecific inhibitor of NOS, significantly attenuated the microvascular leak of lung and kidney and the proteinuria produced by HV t ventilation, which led the investigators to incriminate eNOS as the mediator of injury. Hammerschmidt and coworkers ( 30) also reported an increase in BAL nitrite levels but failed to detect any change in iNOS and eNOS mRNA expression in response to HV t MV in isolated rabbit lungs. A study by Broccard and colleagues ( 31), also in isolated perfused rabbit lungs, demonstrated a positive correlation between NO metabolites in BAL and lung vascular permeability. Finally, a recent study by Lang and coworkers ( 32), in a rabbit model of LPS-induced lung injury, suggests that hypercapnia (produced by changes in ventilator settings) increases lung iNOS expression and amplifies LPS-induced pulmonary inflammatory responses. In none of the above studies was specific NOS activity ever measured. Despite some methodologic differences, the present study is consistent with that of Frank and colleagues ( 11). However, in addition to finding increased iNOS protein expression, we also correlated overall changes in iNOS gene expression and activity to nitrosative stress and capillary permeability. Furthermore, the lack of lung injury, nitrosative stress, or increased capillary permeability in the iNOS-deficient mice strongly suggests a direct role of this NOS isoform in VILI. In relation to the mechanisms of injury, it is unlikely that NO itself is directly responsible for the development of nitrosative damage. NO diffuses quite freely across membranes and its fate is only limited when it encounters hemoglobin, a target enzyme, such as smooth muscle guanylate cyclase ( 3), or less likely, thiol groups to form nitrosothiols (which results in spatial confinement of NO as suggested by Lancaster and Gaston [ 33]) through a tightly regulated cellular process. Different redox forms of NO, such as nitrosonium ion and nitroxyl anion, rather than NO itself, have been considered potential effectors of NOS ( 34). In addition, the fate of the NO molecule will also be limited in the presence of superoxide production (because the reaction of superoxide with NO is much greater than that of superoxide with its scavenger superoxide dismutase), and peroxynitrite will be formed ( 3). We have recently demonstrated that the superoxide-producing enzyme xanthine oxidoreductase is activated by mechanical stress in endothelial cells in vitro (cells subjected to cyclic stretch) and in vivo after exposure of mice to MV ( 35). Therefore, we speculate that lung damage and the resulting capillary permeability demonstrated in the present model are related to iNOS-derived NO reacting with superoxide (from activation of xanthine oxidoreductase or other lung oxidases) to form peroxynitrite in components of the alveolar–capillary membrane. The presence of lung nitrotyrosine (a footprint of peroxynitrite damage) at the site of iNOS increased expression in response to MV supports our contention that iNOS-derived NO is spatially confined, presumably by the local production of superoxide in response to mechanical stress. Regarding potential lung toxicity mediated by NO and its end-products, it is noteworthy that inhaled NO has been used safely and with little evidence of overall toxicity in patients with acute respiratory distress syndrome (ARDS) and patients with pulmonary hypertension. However, there is evidence that inhaled NO causes some nitrotyrosine formation in animal models ( 32, 36) as well as in humans ( 37). Lamb and coworkers ( 37) demonstrated that patients with ARDS receiving inhaled NO had increased levels of 3-nitrotyrosine and 3-chlorotyrosine compared with similar patients not receiving inhaled NO. Formation of 3-nitrotyrosine and 3-chlorotyrosine are presumably mediated by peroxynitrite and possibly interaction between nitrite and hypochlorous acid, respectively. On the basis of these studies, we speculate that the beneficial effect or effects (e.g., improvement in oxygenation) of inhaled NO in patients with ARDS may be mitigated by potential toxicity of this agent, such as formation of nitrotyrosine residues in lung. We further surmise that the net effect of NO (whether from inhaled therapy or cellular source, such as shown in the present study) in the lung depends on a critical balance between NO and superoxide formation, the site(s) and sources of NO release, and associated conditions (e.g., concomitant localized production of superoxide by activated oxidases). The mechanisms of iNOS upregulation in response to MV were not investigated and are beyond the scope of this study. However, several possibilities can be invoked. Type 2 NOS is generally induced by proinflammatory factors, such as interleukins (IL-1, IL-6, and IL-8), TNF-α, and γ-IFN. Animal ( 8) and human ( 6) studies have now shown that some of these cytokines are released in BAL and plasma in response to MV, and that a protective lung strategy (i.e., LV t) can prevent this inflammatory response ( 5, 8). Chemokine and cytokine release may be secondary to activation of nuclear factor–κB ( 38), a transcriptional factor also known to induce iNOS ( 39). To identify mechanical stress–induced candidate genes using schematic representation of cross-species (rat, murine, canine, and human) ortholog database and gene ontology processes, we recently identified inflammatory response ontologies in experimental MV and cell-stretch models. Ontology patterns were heavily represented by several cytokines (e.g., IL-1β and IL-6), cytokine receptors (IL-1 and IL-8 receptors), and chemokines (chemoattractant protein [MCP]-1 and macrophage inflammatory protein [MIP]-2α) in these models ( 40). Using microarray analysis to search for differentially expressed genes in lungs of C57BL/6J mice exposed to 2 hours of MV, we confirmed a dramatic increase in certain cytokines (twofold increase in IL-1β, TNF-α, and γ-IFN, and > 50-fold increase in IL-6) and a twofold increase in iNOS in the present MV model (results not shown). However, we were unable in the present study to detect IL-6, TNF-α, and γ-IFN, even after concentration of the BAL fluid (results not shown), which does not rule out a local release of these cytokines as a mechanism of iNOS induction. Taken together, these results suggest an inflammatory response as the underlying process in iNOS gene activation in response to MV. Although an inflammatory response is potentially responsible for the induction of iNOS, a direct effect of mechanical stress cannot be excluded. There is overwhelming evidence linking cytoskeletal integrity to gene regulation, and a growing interest in iNOS regulation by the cytoskeleton ( 41). Disruption of the actin cytoskeleton upregulates iNOS expression in vascular smooth muscle cells ( 42). Inhibition of Rho proteins (important components of the organization of the cytoskeleton) also upregulates iNOS in epithelial cells through transcriptional and post-transcriptional events ( 43). Therefore, because mechanical stress involves endothelial cytoskeletal reorganization and activation of specific signaling pathways ( 44), we postulate that one potential mechanism for iNOS upregulation in our model is a direct effect of mechanical stress on the cytoskeleton, a possibility currently being tested in our laboratory using cultured pulmonary microvascular endothelial cells subjected to cyclic stretch. Nitric oxide end-products (i.e., nitrite, nitrate, and nitrotyrosine) have been found in BAL of patients with trauma or suspected sepsis, either at risk for or with ARDS, and have been related to increased mortality ( 45). However, although all the patients in this study were on MV, they also had trauma or suspected sepsis. Our study would suggest that MV alone (without any additional trigger of the inflammatory cascade) can upregulate iNOS, thus contributing to formation of NO end-products and nitrosative stress. These changes correlate with increased V t. However, we cannot rule out the possibility of pressure as a stimulus for iNOS induction in view of the significant increase in peak airway pressure in response to HV t ventilation. Nevertheless, it is more likely that volutrauma rather than barotrauma was responsible for the injury itself, as suggested by work by Dreyfuss and colleagues ( 46), using high-pressure/LV t (as provided by thoracoabdominal banding of ventilated rats) and low-pressure/HV t MV, in which lung injury was solely related to changes in lung volume (volutrauma) and not airway pressure (barotrauma). HV t (i.e., 20 ml/kg) was higher in this study compared with our previous study (i.e., 17 ml/kg) demonstrating lung injury in a similar model ( 10), but significantly lower compared with volumes (i.e., 30 ml/kg) reported in the study by Frank and colleagues ( 11) in rats. Although we did not measure neuronal NOS, it is clear that the source of increased NO output in the present study was iNOS and not eNOS (at least for the C57BL/6J mice), as indicated by the changes in mRNA, protein, and respective NOS activities. We were able to identify several potential sources of iNOS by immunohistochemical staining. There was an increase in iNOS immunostaining involving predominantly endothelial but also epithelial cells. We have previously demonstrated that iNOS can be induced by cytokines and hypoxia in cultured pulmonary microvascular endothelial cells, and have postulated a role for this cellular source in endothelial damage (and potential capillary permeability) in inflammatory conditions such as sepsis and ARDS ( 14). We now postulate that these cells can be a significant source of iNOS-derived NO in response to mechanical stress. In that respect, the cellular source of iNOS in our model differs from that of a sepsis-induced ALI after cecal ligation and perforation described by Wang and coworkers ( 47) in which the authors studied iNOS +/+, iNOS −/−, and two reciprocally bone marrow–transplanted iNOS chimeric mice groups (iNOS +/+ donor bone marrow transplanted into iNOS −/− recipient mice and vice-versa) to demonstrate that inflammatory cells accounted for increased iNOS activity and injury. However, previous work from the same group using a similar approach of bone marrow–transplanted chimeric mice incriminated parenchymal cells (similar to the present study) as the predominant source of iNOS activity in LPS-induced lung injury ( 48). Taken together, these studies suggest that the specific source (inflammatory vs. lung parenchymal cells) of iNOS/nitrosative stress in ALI may vary according to the stimulus and mechanism of injury. Mechanical stress–induced capillary permeability has been related to endothelial and epithelial plasma membrane breaks ( 46, 49). However, it is increasingly evident that, aside from mechanical disruption of the alveolar–capillary barrier, certain signaling pathways play a predominant role in the initiation of events leading to formation of intercellular gaps. For instance, inhibitors of myosin light-chain kinase and phosphodiesterase reduce the increased capillary coefficient filtration in animal models of MV-induced injury ( 50). Other signaling pathways altered by MV include tyrosine phosphorylation, focal adhesion formation ( 51), and activation of mitogen-activated protein kinase ( 7). Central to all these pathways is alteration in the cytoskeleton. In that respect, it is worth noting that peroxynitrite causes endothelial barrier dysfunction ( 52). The exact mechanisms involved in endothelial barrier disruption by NO end-products remain to be deciphered and will be best addressed with a combination of in vitro and in vivo studies. In summary, we have shown that mechanical ventilation upregulates lung iNOS gene expression and activity in a mouse model of VILI. In addition, iNOS most likely contributes to ventilator-induced pulmonary capillary leakage through lung nitrosative stress. None of the authors have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. 1. Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–2012. [PubMed] 2. Xie QW, Cho HJ, Calaycay J, Mumford RA, Swiderek KM, Lee TD, Ding A, Troso T, Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992;256:225–228. [PubMed] 3. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol 1996;271:C1424–C1437. [PubMed] 4. Kristof AS, Goldberg P, Laubach V, Hussain SN. Role of inducible nitric oxide synthase in endotoxin-induced acute lung injury. Am J Respir Crit Care Med 1998;158:1883–1889. [PubMed] 5. Acute Respiratory Distress Syndrome Network. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. N Engl J Med 2000;342:1301–1308. [PubMed] 6. Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, Bruno F, Slutsky AS. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized controlled trial. JAMA 1999;282:54–61. [PubMed] 7. Li LF, Yu L, Quinn DA. Ventilation-induced neutrophil infiltration depends on c-Jun N-terminal kinase. Am J Respir Crit Care Med 2004;169:518–524. [PubMed] 8. Tremblay L, Valenza F, Ribeiro SP, Li J, Slutsky AS. Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model. J Clin Invest 1997;99:944–952. [PubMed] 9. Chiumello D, Pristine G, Slutsky AS. Mechanical ventilation affects local and systemic cytokines in an animal model of acute respiratory distress syndrome. Am J Respir Crit Care Med 1999;160:109–116. [PubMed] 10. McVerry BJ, Peng X, Hassoun PM, Sammani S, Simon BA, Garcia JG. Sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury. Am J Respir Crit Care Med 2004;170:987–993. [PubMed] 11. Frank JA, Pittet JF, Lee H, Godzich M, Matthay MA. High tidal volume ventilation induces NOS2 and impairs cAMP-dependent air space fluid clearance. Am J Physiol Lung Cell Mol Physiol 2003;284:L791–L798. [PubMed] 12. Choi WI, Quinn DA, Park KM, Moufarrej RK, Jafari B, Syrkina O, Bonventre JV, Hales CA. Systemic microvascular leak in an in vivo rat model of ventilator-induced lung injury. Am J Respir Crit Care Med 2003;167:1627–1632. [PubMed] 13. Peng X, Birukov KG, Lavoie T, Ma SF, Garcia JG, Hassoun PM. Cyclic stretch upregulates inducible nitric oxide synthase in pulmonary endothelial cells [abstract]. Am J Respir Crit Care Med 2003;167:A117. 14. Zulueta JJ, Sawhney R, Kayyali U, Fogel M, Donaldson C, Huang H, Lanzillo JJ, Hassoun PM. Modulation of inducible nitric oxide synthase by hypoxia in pulmonary artery endothelial cells. Am J Respir Cell Mol Biol 2002;26:22–30. [PubMed] 15. Liao JK, Zulueta JJ, Yu FS, Peng HB, Cote CG, Hassoun PM. Regulation of bovine endothelial constitutive nitric oxide synthase by oxygen. J Clin Invest 1995;96:2661–2666. [PubMed] 16. Peng X, Hassoun PM, Sammani S, McVerry BJ, Burne MJ, Rabb H, Pearse D, Tuder RM, Garcia JG. Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 2004;169:1245–1251. [PubMed] 17. Patterson CE, Rhoades RA, Garcia JG. Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol 1992;72:865–873. [PubMed] 18. Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L, Kasahara Y, Salvemini D, Voelkel NF, Flores SC. Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade. Am J Respir Cell Mol Biol 2003;29:88–97. [PubMed] 19. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, Le Cras TD, Abman S, Hirth PK, Waltenberger J, Voelkel NF. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 2000;106:1311–1319. [PubMed] 20. Bredt DS, Snyder SH. Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci USA 1990;87:682–685. [PubMed] 21. Ungureanu-Longrois D, Balligand JL, Okada I, Simmons WW, Kobzik L, Lowenstein CJ, Kunkel SL, Michel T, Kelly RA, Smith TW. Contractile responsiveness of ventricular myocytes to isoproterenol is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture. Circ Res 1995;77:486–493. [PubMed] 22. Sade K, Schwartz I, Schwartz D, Wolman Y, Chernichovski T, Fireman E, Iaina A, Kivity S. Effect of montelukast pretreatment on inducible nitric oxide synthase mRNA expression in the lungs of antigen-challenged allergic mice. Clin Exp Allergy 2003;33:1741–1746. [PubMed] 23. Okawa T, Asano K, Takahashi H, Hashimoto S, Anbe H, Sato A, Gafield RE. Expression of iNOS mRNA and inhibitory effect of NO on uterine contractile activity in rats are determined by local rather than systemic factors of pregnancy. J Pharmacol Sci 2004;95:349–354. [PubMed] 24. Gosgnach W, Messika-Zeitoun D, Gonzalez W, Philipe M, Michel JB. Shear stress induces iNOS expression in cultured smooth muscle cells: role of oxidative stress. Am J Physiol Cell Physiol 2000;279:C1880–C1888. [PubMed] 25. Watanuki M, Sakai A, Sakata T, Tsurukami H, Miwa M, Uchida Y, Watanabe K, Ikeda K, Nakamura T. Role of inducible nitric oxide synthase in skeletal adaptation to acute increases in mechanical loading. J Bone Miner Res 2002;17:1015–1025. [PubMed] 26. Ziegler T, Silacci P, Harrison VJ, Hayoz D. Nitric oxide synthase expression in endothelial cells exposed to mechanical forces. Hypertension 1998;32:351–355. [PubMed] 27. Kuebler WM, Uhlig U, Goldmann T, Schael G, Kerem A, Exner K, Martin C, Vollmer E, Uhlig S. Stretch activates nitric oxide production in pulmonary vascular endothelial cells in situ. Am J Respir Crit Care Med 2003;168:1391–1398. [PubMed] 28. Erwin PA, Lin AJ, Golan DE, Michel T. Receptor-regulated dynamic S-nitrosylation of endothelial nitric oxide synthase in vascular endothelial cells. J Biol Chem 2005;280:19884–19894. 29. Quinn DA, Moufarrej RK, Volokhov A, Hales CA. Interactions of lung stretch, hyperoxia, and MIP-2 production in ventilator-induced lung injury. J Appl Physiol 2002;93:517–525. [PubMed] 30. Hammerschmidt S, Schiller J, Kuhn H, Meybaum M, Gessner C, Sandvoss T, Arnold K, Wirtz H. Influence of tidal volume on pulmonary NO release, tissue lipid peroxidation and surfactant phospholipids. Biochim Biophys Acta 2003;1639:17–26. [PubMed] 31. Broccard AF, Feihl F, Vannay C, Markert M, Hotchkiss J, Schaller MD. Effects of L-NAME and inhaled nitric oxide on ventilator-induced lung injury in isolated, perfused rabbit lungs. Crit Care Med 2004;32:1872–1878. [PubMed] 32. Lang JD, Figueroa M, Sanders KD, Aslan M, Liu Y, Chumley P, Freeman BA. Hypercapnia via reduced rate and tidal volume contributes to lipopolysaccharide-induced lung injury. Am J Respir Crit Care Med 2005;171:147–157. [PubMed] 33. Lancaster JR Jr, Gaston B. NO and nitrosothiols: spatial confinement and free diffusion. Am J Physiol Lung Cell Mol Physiol 2004;287:L465–L466. [PubMed] 34. Schmidt HH, Hofmann H, Schindler U, Shutenko ZS, Cunningham DD, Feelisch M. No .NO from NO synthase. Proc Natl Acad Sci USA 1996;93:14492–14497. [PubMed] 35. Abdulnour RE, Peng X, Han EJ, Hasan EJ, Kayyali U, Garcia JG, Hassoun PM. Mechanical stress upregulates xanthine oxidoreductase through a p38 MAP kinase-dependent pathway [abstract]. Am J Respir Cell Mol Biol 2005;2:A717. 36. Weinberger B, Fakhrzadeh L, Heck DE, Laskin JD, Gardner CR, Laskin DL. Inhaled nitric oxide primes lung macrophages to produce reactive oxygen and nitrogen intermediates. Am J Respir Crit Care Med 1998;158:931–938. [PubMed] 37. Lamb NJ, Quinlan GJ, Westerman ST, Gutteridge JM, Evans TW. Nitration of proteins in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome receiving inhaled nitric oxide. Am J Respir Crit Care Med 1999;160:1031–1034. [PubMed] 38. Held HD, Boettcher S, Hamann L, Uhlig S. Ventilation-induced chemokine and cytokine release is associated with activation of nuclear factor-kappaB and is blocked by steroids. Am J Respir Crit Care Med 2001;163:711–716. [PubMed] 39. Marks-Konczalik J, Chu SC, Moss J. Cytokine-mediated transcriptional induction of the human inducible nitric oxide synthase gene requires both activator protein 1 and nuclear factor kappaB-binding sites. J Biol Chem 1998;273:22201–22208. [PubMed] 40. Grigoryev DN, Finigan JH, Hassoun P, Garcia JG. Science review: searching for gene candidates in acute lung injury. Crit Care 2004;8:440–447. [PubMed] 41. Marczin N, Jilling T, Papapetropoulos A, Go C, Catravas JD. Cytoskeleton-dependent activation of the inducible nitric oxide synthase in cultured aortic smooth muscle cells. Br J Pharmacol 1996;118:1085–1094. [PubMed] 42. Hattori Y, Kasai K. Disruption of the actin cytoskeleton up-regulates iNOS expression in vascular smooth muscle cells. J Cardiovasc Pharmacol 2004;43:209–213. [PubMed] 43. Witteck A, Yao Y, Fechir M, Forstermann U, Kleinert H. Rho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression. Exp Cell Res 2003;287:106–115. [PubMed] 44. Birukov KG, Jacobson JR, Flores AA, Ye SQ, Birukova AA, Verin AD, Garcia JG. Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch. Am J Physiol Lung Cell Mol Physiol 2003;285:L785–L797. [PubMed] 45. Sittipunt C, Steinberg KP, Ruzinski JT, Myles C, Zhu S, Goodman RB, Hudson LD, Matalon S, Martin TR. Nitric oxide and nitrotyrosine in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;163:503–510. [PubMed] 46. Dreyfuss D, Soler P, Basset G, Saumon G. High inflation pressure pulmonary edema: respective effects of high airway pressure, high tidal volume, and positive end-expiratory pressure. Am Rev Respir Dis 1988;137:1159–1164. [PubMed] 47. Wang LF, Patel M, Razavi HM, Weicker S, Joseph MG, McCormack DG, Mehta S. Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 2002;165(12):1634–1639. [PubMed] 48. Wang LF, Mehta S, Weicker S, Scott JA, Joseph M, Razavi HM, McCormack DG. Relative contribution of hemopoietic and pulmonary parenchymal cells to lung inducible nitric oxide synthase (iNOS) activity in murine endotoxemia. Biochem Biophys Res Commun 2001;283:694–699. [PubMed] 49. West JB, Tsukimoto K, Mathieu-Costello O, Prediletto R. Stress failure in pulmonary capillaries. J Appl Physiol 1991;70:1731–1742. [PubMed] 50. Parker JC. Inhibitors of myosin light chain kinase and phosphodiesterase reduce ventilator-induced lung injury. J Appl Physiol 2000;89:2241–2248. [PubMed] 51. Bhattacharya S, Sen N, Yiming MT, Patel R, Parthasarathi K, Quadri S, Issekutz AC, Bhattacharya J. High tidal volume ventilation induces proinflammatory signaling in rat lung endothelium. Am J Respir Cell Mol Biol 2003;28:218–224. [PubMed] 52. Knepler JL Jr, Taher LN, Gupta MP, Patterson C, Pavalko F, Ober MD, Hart CM. Peroxynitrite causes endothelial cell monolayer barrier dysfunction. Am J Physiol Cell Physiol 2001;281:C1064–C1075. [PubMed]
|
This article has been 
