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TRANSLATIONAL PHYSIOLOGY

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways

¹Division of Pulmonary and Critical Care Medicine, ²Department of Medicine, Department of Environmental Health Sciences, Bloomberg School of Public Health, and ³Division of Cardiopulmonary Pathology and Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and ⁴Pulmonary and Critical Care Division, Tupper Research Institute, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts

Submitted 27 October 2005 ; accepted in final form 7 April 2006

ABSTRACT

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.

mechanical ventilation; acute lung injury; mitogen-activated protein kinase

The enzyme xanthine oxidoreductase (XOR) is an important component of an integrated inflammatory response in organ dysfunction (14) and has been implicated in the pathogenesis of ARDS because of its ability to generate reactive oxygen species (ROS). We postulated that XOR might be upregulated by mechanical stress and might contribute to the development of VILI. Because XOR activation in hypoxia occurs through p38 MAP kinase activation (18), we tested activation of the different MAP kinase pathways in response to mechanical stress in vivo and in vitro and their effects on XOR expression. Our results indicate that XOR activity is rapidly upregulated both in vivo and in vitro through activation of ERK and p38 MAP kinase. Furthermore, allopurinol prevented pulmonary vascular leakage produced by HT_V MV, suggesting that XOR contributes to increased capillary permeability produced by mechanical stress in this model. Some of the results of these studies have been reported previously in the form of an abstract (29).

MATERIALS AND METHODS

Materials and reagents. RPMI 1640 and fetal bovine serum were obtained from Hyclone (Logan, UT). Dithiothreitol (DTT), CHAPS, EDTA, PMSF, leupeptin, methylene blue, allopurinol, and SB-203580 were obtained from Sigma (St. Louis, MO). PD-98059 and U0126 were obtained from Calbiochem (San Diego, CA). Phospho-specific antibodies directed at p38 MAP kinase, ERK1/2, ERK5, and JNK, along with anti-total MAP kinase antibodies, were obtained from Cell Signaling Technology (Beverly, MA). Anti-XOR antibody was obtained from NeoMarkers (Fremont, CA).

Experimental protocol and animal exposure to MV. Male C57BL/6J mice aged 7–10 wk (Jackson Laboratory, Bar Harbor, ME) were studied in a pathogen-free facility under a protocol approved by the Johns Hopkins Department of Laboratory Animal Medicine. Animals were first anesthetized with intraperitoneal ketamine (150 mg/kg) and acetylpromazine (15 mg/kg). A neck midline incision was then performed for exposure of the trachea to facilitate endotracheal intubation of the mouse with a 20-gauge 12-in.-long catheter (Johnson and Johnson, New Brunswick, NJ). The animals were then subjected to MV (Harvard Apparatus, Boston, MA) with room air for 0 (sham), 0.5, 1, or 2 h with LV_T (7 ml/kg) and HV_T (20 ml/kg). The respiratory rate (RR) was set at 110 breaths/min for all tidal volumes, and the dead space was adjusted to maintain arterial pH between 7.35 and 7.45. Airway pressures continuously measured during MV at 7 and 20 ml/kg revealed that end-expiratory pressures remained 0–2 cmH₂O throughout the 2-h period for both LV_T and HV_T. Mean blood pressure was continuously monitored via tail cuff using a blood pressure monitor (BP-1) and a data acquisition system (World Precision Instruments, Sarasota, FL) and remained typically 85 mmHg. The adequacy of MV settings on gas exchange was confirmed in preliminary experiments in which arterial blood gases obtained via catheterization of a femoral artery and analyzed by an automated blood gas analyzer (Instrumentation Laboratories, Lexington, MA) revealed stable levels of arterial oxygen (Pa_O2 of 65–80 mmHg) and carbon dioxide (Pa_CO2 of 30–40 mmHg). At the end of MV, the animals were administered an intraperitoneal lethal dose of the anesthetic agent before the lungs were harvested.

Drug delivery. To assess the role of XOR on MV-induced pulmonary vascular permeability, mice received in some experiments a single dose of allopurinol (50 mg/kg) by gavage or a similar volume of vehicle (saline) 16 h before exposure to MV. In vivo efficiency of the drug dosage in inhibiting XOR activity was confirmed in preliminary experiments.

Assessment of pulmonary capillary permeability. Evans blue dye (EBD, 20 ml/kg) was injected into the external jugular vein 30 min before termination of the experiment to assess vascular leak as previously described (28, 30). In brief, at the end of the experimental protocol, a thoracotomy was performed, and the lungs were perfused free of blood with PBS containing 5 mM EDTA before being excised en bloc, blotted dry, weighed, and snap frozen in liquid nitrogen. The right lung was homogenized in phosphate-buffered saline (PBS, 1 ml/100 µg tissue), incubated with 2 vol formamide (18 h, 60°C), and centrifuged at 5,000 g for 30 min, and the optical density of the supernatant was determined by spectrophotometry at 620 nm. Extravasated EBD concentration (µg EBD per lung) in lung homogenates was calculated against a standard curve as previously described (23).

Bronchoalveolar lavage protein concentration. Bilateral bronchoalveolar lavage (BAL) was performed with 1 ml of saline for determination of BAL fluid protein concentration as previously described (13).

Processing of lung tissue for immunohistochemical staining. At the end of experimental exposure, the right external jugular vein was catheterized, and the lungs were flushed free of blood with ice-cold PBS containing the phosphatase inhibitor sodium orthovanadate (1 µM). The lungs were inflated to 25 cmH₂O with 0.2% of low-melting agarose, harvested, and then stored in 10% formalin for 14 h before being embedded in paraffin. After deparaffinization, tissue sections were incubated for 1 h at room temperature with a phospho-specific p38 antibody (1:150), followed by horseradish peroxidase-conjugated secondary antibody. After several washes with PBS, the immunohistochemical reaction was visualized by incubation with 0.05% diaminobenzidine containing 0.01% H₂O₂ in 50 mM Tris (pH 7.6).

Exposure of endothelial cells to cyclic stretch. Rat pulmonary microvascular endothelial cells (RPMECs) were cultured as previously described (8). All experiments involving cyclic stretch (CS) were performed as previously described (5), using a FX-4000T Flexercell Tension Plus system (Flexcell International, McKeesport, PA) equipped with a 25-mm BioFlex loading station designed to provide uniform radial and circumferential strain across the membrane surface of loaded Bioflex plates. All CS experiments were performed in the presence of complete culture medium containing 10% fetal bovine serum. RPMECs were seeded at standard densities (4 x 10⁵ cells/plate) onto collagen I-coated flexible-bottomed BioFlex plates, whether for the static group (control) or the stretched group, to ensure standard culture conditions. Culture medium was changed every other day until cells reached confluence. The plates were then loaded onto the Flexercell system and exposed to CS for 2 h at 20 cycles per minute and 18% elongation. BioFlex plates with control endothelial cells exposed to static conditions were placed in the same incubator as stretched plates. When necessary, RPMECs were preincubated with a p38 inhibitor (1 µM SB-203580), ERK inhibitors (PD-98059 or U0126), or similar amounts of vehicle (DMSO) for 1 h before CS. At the end of the experiment, cells were lysed in an XOR lysis buffer consisting of 50 mM sodium phosphate (pH 7.4), 1.5 mg/ml DTT, 0.1 mM EDTA, 1 mg/ml CHAPS, 0.18 mg/ml PMSF, and 0.5 µg/ml leupeptin.

XOR activity measurement. The activities of xanthine dehydrogenase (XDH) and xanthine oxidase (XO) in response to different treatments were assayed in cells and lung homogenates using a fluorimetric assay that measured both XO and XDH activities, as previously described (3). The principle of the assay involves the conversion of pterin into the fluorescent product isoxanthopterin. The rate of product formation with oxygen as the electron acceptor represents the activity of XO, whereas the combined activities of XO and XDH are measured with methylene blue as the electron acceptor. In brief, cells were washed once in PBS and then scraped off the plate in XOR buffer. The cells were sonicated for 5 s and centrifuged at 10,000 g for 5 min. The supernatant was collected and assayed immediately or stored at –80°C overnight. XOR activity was adjusted for micrograms of protein per milliliter of lysate and then normalized to control XOR activity.

For determination of lung XOR activity, the left lung was harvested and homogenized in XOR lysis buffer. We then centrifuged the homogenate for 20 min at 15,000 rpm before measuring activity as described above. Because changes in XO and XDH occurred in parallel in response to mechanical stress, results are expressed in all experiments as total XOR activity.

SDS-PAGE and immunoblot analysis. Aliquots from cell lysates and tissue homogenates prepared as described above were assayed for protein measurement using the Bradford protein assay (12) and then diluted with Laemmli loading buffer for SDS-PAGE (13). Equal amounts of protein were then loaded in each well of 4–20% Tris-glycine gels. After electrophoresis for 90 min at 125 V of constant voltage, the gel was blotted onto a polyvinylidene difluoride membrane by electrophoretic transfer at 25 V of constant voltage for 1 h. The membrane was then washed, blocked with 5% blocking solution, and probed with antibodies against XOR and MAP kinases and their phosphorylated forms. The immunoreactive bands were visualized using a secondary antibody conjugated to horseradish peroxidase and a chemiluminescent detection system (ECL; Amersham, Piscataway, NJ). Films were scanned using a Color Imager Scanner (Seiko Epson, Tokyo, Japan) and National Institutes of Health Image 1.44 software.

Semiquantitative RT-PCR. Total RNA was isolated using a commercially available kit (Qiagen, Valencia, CA) and then quantified and loaded in an RT-PCR mix for a one-step semiquantitative RT-PCR amplification (Invitrogen, Carlsbad, CA). The PCR reaction was carried out in a 25-µl solution. The pair of sense and antisense primers specific for mouse and rat XOR, designed based on published cDNA sequences, was as follows: 5'-AGGTCGCCATAACCTGTGGGCTG-3' (forward primer) and 5'-ATTGAGGTCAGCACTGGCAGAGG-3' (reverse primer) (19). After 30 cycles, 15 µl of PCR products were analyzed by gel electrophoresis on a 1% agarose gel and visualized by ethidium bromide staining. To assess the adequacy of the cDNA and the efficiency of the RT-PCR system, primers for 18S rRNA were used as RT-PCR controls under the same conditions. The cDNA for 18S rRNA was amplified with the following primers: 5'-CGGCTACCACATCCAAGGAA-3' (forward) and 5'-GCTGGAATTACCGCGGCT-3' (reverse). The steady-state mRNA levels are expressed in arbitrary units as the ratio of XOR/18S expression (25).

Statistics. Values are shown as means ± SD, with n 4 for each experimental condition. Data were analyzed by a one-way ANOVA with Bonferroni correction. Significance in all cases was defined as P < 0.05.

RESULTS

MV stimulates XOR activity. We investigated the effects of MV on changes in XOR expression and activity in C57BL/65 mice breathing spontaneously (sham) or exposed to ventilation with LV_T or HV_T for 2 h. Although the animals received different tidal volumes, they were ventilated at the same RR with adjustment in dead space (as detailed in MATERIALS AND METHODS). At the end of exposure, lungs were removed and XOR protein expression and activity were assessed as described in MATERIALS AND METHODS.

As shown in Fig. 1, MV with HV_T resulted in a significant increase (by ANOVA followed by Bonferroni correction) in lung XOR activity compared with LV_T or sham animals (1.7-fold increase with HV_T, P < 0.05).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Mechanical ventilation (MV) induces xanthine oxidoreductase (XOR) enzymatic activity. C57BL/6J mice were randomly exposed to spontaneous breathing (sham) or to MV at low (LVT) or high tidal volume (HVT) for 2 h. The lungs were then harvested and homogenized, and XOR activity was measured as described in MATERIALS AND METHODS. XOR activity was significantly upregulated in response to ventilation with HVT compared with sham or LVT. *P < 0.01 vs. control or LVT; n = 7–10 mice/group. There was no significant change in XOR activity in response to ventilation with LVT.

View larger version (20K): [in this window] [in a new window]   Fig. 2. XOR activation is mediated by posttranslational modification. Lungs from mice ventilated with HVT for 2 h were homogenized, and both RNA and protein were isolated for subsequent semiquantitative RT-PCR and immunoblotting. A: repeated assays showed no increase in XOR mRNA when adjusted for the internal standard 18S rRNA. B: similarly, immunoblotting using a specific XOR antibody showed no increase in protein level compared with the internal standard -actin. C: densitometric analysis of the immunoblots confirmed the absence of increased XOR protein (n = 3 for each condition).

View larger version (55K): [in this window] [in a new window]   Fig. 3. Activation of MAP kinases (MAPKs) by MV. C57BL/6J mice were exposed to spontaneous breathing (sham) or to 30, 60, and 120 min of MV with LVT and HVT. The animals were then killed, and lungs were either homogenized in lysis buffer for immunoblotting using antibodies directed at the different phospho-MAPKs or fixed in agarose followed by paraffin for immunohistochemistry studies. A: MV with LVT (left) does not result in MAPK phosphorylation. However, immunoblots from mice ventilated at HVT (right) demonstrate an increase in p38 MAPK, ERK1/2, and ERK5 phosphorylation as early as 60 min after initiation of MV, with no significant change in JNK phosphorylation. p, Phosphorylated; t, total. B, A: representative immunohistochemistry slide from a sham animal shows no phospho-p38 MAPK immunostaining. B, B: lung from a mouse exposed to ventilation with HVT shows diffuse phospho-p38 immunoreactivity, as well as nuclear distribution in both endothelial and epithelial cells.

Confluent RPMECs were exposed to static conditions (no stretch) or CS (20 cycles/min and 5 or 18% elongation) for 1 and 2 h before measurements of intracellular XOR activity. CS at 5% did not alter XOR activity at 1 (Fig. 4) or 2 h (not shown). However, CS at 18% resulted in a significant upregulation of XOR activity at 1 and 2 h of stretch (1.53 ± 0.4- and 1.59 ± 0.11-fold increase, respectively, P < 0.05) compared with static conditions (Fig. 4).

View larger version (10K): [in this window] [in a new window]   Fig. 4. Cyclic stretch (CS) induces XOR enzymatic activity. Rat pulmonary microvascular endothelial cells (RPMVECs) grown on collagen I-coated BioFlex plates were kept in static conditions (control) or subjected to CS at 5 and 18% elongation and 20 cycles/min for 1 and 2 h (for 18% elongation only). Cells were then collected, and XOR activity was measured. Although CS at 5% elongation did not have any effect, it significantly upregulated XOR enzymatic activity at 1 and 2 h at 18% elongation compared with control. *P < 0.05 vs. control; n = 5–8 six-well plates/group.

View larger version (16K): [in this window] [in a new window]   Fig. 5. XOR activation by CS is not transcriptionally mediated. Semiquantitative RT-PCR was performed on RNA isolated from stretched cells as described in MATERIALS AND METHODS. Repeated runs showed no increase in XOR mRNA compared with the internal standard 18S rRNA.

View larger version (97K): [in this window] [in a new window]   Fig. 6. MAPK activation by CS. RPMVECs were exposed to 0, 5, 30, 60, and 120 min of CS before immunoblotting of cell lysates using antibodies against t- and p-MAPKs. There was increased phosphorylation of p38, ERK1/2, and ERK5 as early as 5 min of CS. However, there was no evidence of JNK phosphorylation (not shown).

As shown in Fig. 7, the two drugs had somewhat opposite profiles. PD-98059 inhibited ERK5 phosphorylation at all doses but significantly prevented ERK1/2 activation only at a dose of 100 µM. On the other hand, U0126 prevented ERK1/2 activation at doses as low as 0.3 µM while inhibiting ERK5 phosphorylation only at the maximal dose of 10 µM. Therefore, for the following experiments, U0126 (1 µM) and PD-98095 (10 µM) were used to inhibit ERK1/2 and ERK5, respectively. There was no evidence of cellular toxicity with these doses as assessed by phase-contrast microscopy or trypan blue exclusion.

View larger version (32K): [in this window] [in a new window]   Fig. 7. Effect of MAPK inhibitors on CS-induced ERK activation. RPMVECs were exposed to 10 min of CS after 1 h pretreatment with increasing doses of U0126 and PD-98059, 2 MAPK cascade inhibitors. There was significant ERK1/2 inhibition with no effect on ERK5 activation with 0.3–3 µM U0126, whereas there was no effect on ERK1/2 activation but significant blunting of ERK5 phosphorylation with 10 µM PD-98059.

View larger version (14K): [in this window] [in a new window]   Fig. 8. Treatment with p38 and ERK inhibitors prevent XOR upregulation by CS. RPMECs were exposed to CS for 1 h in the presence of the specific p38 MAPK inhibitor SB-203580 (1 µM, added 1 h before stretch), the ERK1/2 inhibitor U0126 (1 µM), the ERK5 inhibitor PD-98095 (10 µM), a combination of U0126 (1 µM) and PD-98095 (10 µM), or vehicle (DMSO). There was a significant increase in XOR activity in response to CS, which was significantly prevented by pretreatment with SB-203580, PD-98095, U0126, or the combination of PD-98095 and U0126. *P < 0.05 vs. static control; +P < 0.05 vs. stretch with any treatment.

There was no significant change in BAL protein concentration and in EBD lung content in animals exposed to LV_T mice compared with sham control animals (data not shown). However, there was a significant increase in both BAL protein concentration (1.6-fold increase; P < 0.05, Fig. 9A) and EBD extravasation (1.55-fold increase; P < 0.05, Fig. 9B) in mice exposed to HV_T compared with sham controls. Allopurinol treatment significantly attenuated the increase in BAL protein and EBD accumulation in mice exposed to HV_T (Fig. 9, A and B) while almost completely inhibiting XOR activity (Fig. 9C), strongly suggesting a significant role for XOR in the pathogenesis of ventilator-induced pulmonary edema.

View larger version (12K): [in this window] [in a new window]   Fig. 9. Allopurinol prevents ventilation-induced pulmonary capillary leakage. C57BL/6J mice were randomly exposed to spontaneous breathing (sham) or to MV with HVT for 2 h with and without allopurinol pretreatment. A: bronchoalveolar lavage (BAL) fluid revealed significantly increased protein concentration in the ventilated group compared with sham, a finding that was prevented by allopurinol treatment. B: in separate experiments, Evans blue dye (EBD) was administered through the right jugular vein 30 min before the termination of the experiment. Analysis of lung homogenates revealed increased EBD in ventilated lungs. C: treatment with allopurinol (Allo, 50 mg/kg) completely inhibited lung XOR activity. *P < 0.05 vs. sham; P < 0.05 vs. allopurinol treatment in same group.

XOR, best known for its role in purine catabolism and as a target in gout therapy, exists as two interconvertible forms, XDH and XO. The conversion of XDH to XO can be reversible, after treatment with sulphide reagents, or irreversible, after proteolysis (7). In the process of oxidation of hypoxanthine to uric acid, NAD⁺ and molecular oxygen are the preferential electron acceptors for XDH and XO, respectively. This relates to the inability of NAD⁺ to bind to XO due to significant conformational changes of the enzyme. Therefore, conversion of XDH to XO by either proteolysis or PTM significantly increases the amount of ROS produced by XOR (34, 38). However, reduction of molecular oxygen by either form of the enzyme yields superoxide and H₂O₂, and upregulation of overall XOR activity, irrespective of XDH-to-XO ratios, can lead to increased ROS levels (14). It is the capacity of XOR to generate such ROS that is of major interest in clinical syndromes.

The consequences of ROS release following the induction of XOR have been implicated in the pathogenesis of several diseases such as ischemia-reperfusion injury (21), multisystem organ dysfunction syndrome (12), and cardiovascular diseases (4). The contribution of XOR to acute lung injury has also been demonstrated (11, 31, 37). Although it is generally thought that XOR is induced by cytokines, inflammatory products, and hypoxia, to date, there is no evidence supporting regulation of this enzyme by mechanical forces in the lung or its contribution to VILI. However, we have recently shown in a similar murine model of VILI that MV results in peroxynitrite (ONOO^–) formation as evidenced by nitrotyrosine deposition in the lung (29). Because ONOO^– formation requires superoxide production, we sought to determine whether cyclic mechanical stress induced by MV could alter XOR activity and whether the activation of XOR contributed to the development of capillary permeability related to VILI.

This study demonstrates for the first time that lung XOR enzymatic activity is increased by mechanical stress. Our animal model of MV demonstrates that HV_T ventilation causes an increase in lung XOR activity compared with spontaneous ventilation or LV_T MV considered clinically safe in humans (i.e., 7 ml/kg). In addition, these studies demonstrate evidence of VILI in our animal model as evidenced by increasing BAL total protein concentration and lung EBD extravasation, both reflecting alveolar-capillary barrier dysfunction. The XOR inhibitor allopurinol prevented the increase in capillary pulmonary permeability produced by MV with HV_T, which strongly suggests that XOR activation by mechanical stress contributes to the ventilator-induced alveolar barrier dysfunction.

The injury in this model of VILI seems to be characterized by increased permeability of the epithelial-endothelial barrier without measurable inflammation. The efflux of protein into the alveolar space, which is reflected by the increase in BAL protein indicates substantial epithelial injury, whereas the EBD extravasation that accompanies MV suggests injury to the endothelium. On the other hand, the evaluation of BAL inflammatory cells showed no evidence of neutrophil infiltration (data not shown). These findings are in contrast with a recent report of a VILI model where we demonstrated evidence of neutrophil infiltration and unchanged BAL protein concentration (29). However, the two MV models differ by the RR used; we speculate that ventilating at the higher rate of 110 beats/min as in the current study minimizes the inflammatory reaction while maximizing alveolar barrier dysfunction. We also suspect that the more consistent endothelial and epithelial damage obtained in the current model is related to repetitive opening and closing of the alveoli at a higher RR (110 beats/min as opposed to 60 beats/min for the previous study).

XOR is widely distributed among species and within various tissues (2), with abundant concentrations in the lung (27). Immunofluorescence and immunoperoxidase techniques have allowed the localization of XOR in the cytoplasm of endothelial cells in capillaries of several organs including the lung (17), indicating that XOR is essentially expressed in vascular endothelial cells (17). Therefore, we explored the response of cultured pulmonary microvascular endothelial cells to CS. We opted for an 18% elongation based on the knowledge that this extent of linear distension corresponds roughly to a 35–50% increase in cell surface area, which is relevant to pathophysiological conditions produced by MV (5). Our in vitro model confirms activation of XOR by mechanical stress in the absence of other confounding factors, such as paracrine interactions from neighboring cells (e.g., macrophages or neutrophils). Mirroring the in vivo findings, no upregulation of XOR gene expression in cultured cells was found.

In regard to the mediators of injury, we have recently demonstrated that the pulmonary leak produced by mechanical stress in response to MV is at least partially related to oxidative/nitrosative stress. Indeed, inducible nitric oxide (NO) synthase (iNOS) is activated by mechanical stress in response to MV, and there is increased nitrotyrosine deposition, a footprint of ONOO^– damage (29). Using fluorescent immunolocalization, we demonstrated that peroxynitirite formation occurs at the site of increased iNOS expression, i.e., essentially in endothelial cells. Furthermore, the lack of nitrotyrosine formation in iNOS-deficient mice allowed us to directly incriminate iNOS upregulation in nitrosative stress. ONOO^–, a powerful oxidant, results from the rapid reaction between NO and superoxide. Because capillary permeability is prevented by iNOS deficiency or pharmacological inhibition of either iNOS (29) or XOR (as in the present study), we postulate that damage to the endothelial barrier is related to endothelial XOR-derived ROS reacting locally with iNOS-derived NO to form ONOO^– in components of the alveolar-capillary membrane.

In relation to XOR expression, the lack of protein upregulation in both our animal and cellular models suggests a PTM as a mechanism of activation. Having demonstrated the role of p38 MAP kinase in XOR PTM and activation by hypoxia (18), we sought to determine the contribution of the MAP kinases to the MV-related XOR activation. Both ERK1/2 and p38 were activated by MV and CS, respectively, confirming findings from our group (5) and others (35). In addition, we now demonstrate that ERK5, a recently discovered MAP kinase with a role in many disease processes (16), is activated by mechanical stress. Furthermore, we demonstrate using pharmacologic inhibitors that both p38 and ERKs are involved in XOR activation by CS. A potential limitation of our study lies in the general lack of specificity of pharmacologic inhibitors, which can be obviated by targeting MAP kinase effectors using gene knockout or silencing with short interfering RNA. These confirmatory experiments are currently being developed in our laboratory. The absence of JNK activation in our model does not preclude a role for this effector in VILI, a finding that has been suggested by other investigators (20). The absence of MAP kinase activation in response to LV_T is likely related to lack of significant alveolar overdistension as this volume is closer to the murine normal tidal volume.

The mechanisms by which the addition of phosphate groups, whether in response to hypoxia (9) or mechanical stress, leads to increased XOR activity need further investigation and are beyond the scope of the current study. However, one might speculate that the negative charges in the XOR molecule introduced by phosphorylation might affect the affinity of the enzyme for its substrates (24). Alternatively, the phosphorylation of XOR might alter its subcellular localization and activity by modulating its interaction with other regulatory proteins.

In summary, this study demonstrates MAP kinase-dependent XOR activation by mechanical stress and subsequent injury in a model of VILI. The possibility of XOR as a downstream target of signaling pathways involved in inflammation and stress responses lends support to the role of XOR activation as an early cellular response to a variety of stressors (36), such as hypoxia (18), LPS (15), and mechanical stress. The availability of highly effective pharmacological inhibitors of XOR with outstanding clinical safety profiles emphasizes the relevance of the present findings. However, the effectiveness of such inhibitors in minimizing the iatrogenic effects of MV in humans with complex medical conditions remains to be studied.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01 HL-049441 and P50 HL-73994.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the expertise and advice provided by Julie Nijmeh, Rachel Damico, and Ben Ho Park. The authors also thank Ellen G. Reather for expert manuscript preparation.

FOOTNOTES

Address for reprint requests and other correspondence: P. M. Hassoun, Div. of Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (e-mail: phassoun{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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