Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury.
- acute lung injury
- nitric oxide
- intercellular adhesion molecule
mechanical ventilation is presently a therapeutic mainstay in the critically ill patients with acute lung injury and acute respiratory distress syndrome. It is now well recognized from animal experiments and increasing evidence in humans that mechanical ventilation per se can cause further lung injury and perhaps lead to the development of multiple organ failure (11). Ventilator-induced lung injury (VILI) as a result of mechanical stretch is characterized by massive inflammatory responses in the lung as evidenced by alveolar-capillary hyperpermeability, protein-rich edema formation, and polymorphonuclear neutrophil (PMN) infiltration into lung interstitium and alveoli (2, 11).
Calpains are Ca2+ dependent, nonlysosomal cysteine proteases (proteolytic enzymes) (12). Several calpain isoforms, including calpain 1 (or μ-calpain), calpain 2 (or m-calpain), and calpain 10, are ubiquitously expressed in mammals, whereas calpain 3 (muscle p94) and calpain 8 (stomach nCl-2) reveal tissue-specific expression patterns (25). Calpain 1 and calpain 2, the two major isoforms of calpain, are heterodimers comprised of a unique large (80 kDa) catalytic subunit and a common small (28 kDa) regulatory subunit. Calpain 1 and calpain 2 require micromolar (1 to 20 μM) and millimolar (0.25 to 0.75 mM) concentration of intracellular Ca2+ for activation (25), respectively. Elevations in intracellular Ca2+ above isoform-specific thresholds induce Ca2+ binding and subsequent activation via a conformational change that creates an active catalytic triad (14), as well as autolysis of both subunits. Calpain 1-deficient mice have a platelet dysfunction (5), whereas knocking down both calpain 1 and 2 simultaneously leads to an embryonic-lethal phenotype (3). The physiological functions of calpains are not yet fully elucidated. Transient calpain activation has been shown to be involved in essential functions such as cell signaling and protein turnover (12), whereas sustained activation of calpains contributes to acute neurological injuries and Alzheimer's disease (25). Recently, calpains have been implicated in apoptotic cell death and tissue injury and appear to be an essential regulator of inflammation (7, 17, 27). During sepsis, calpain 1 has been shown to induce apoptosis in pulmonary microvascular endothelial cells (17). In addition, inhibition of calpains attenuated zymosan-induced multiple organ failure (7), the activation of NF-κB, and organ injury in hemorrhagic shock in the rat (27).
In the present study, using genetic and pharmacological approaches, we identified a crucial role for calpains in the pathogenesis of ventilator-induced lung inflammation and injury. Rapid calpain activation following mechanical stretch triggers PMN infiltration into the lung via endothelial nitric oxide (NO) synthase (eNOS or NOS-3)-NO-mediated ICAM-1 phosphorylation. Our data suggest that calpains may be a key mechanical sensor of early lung inflammation and injury.
MATERIALS AND METHODS
All studies were approved by the University of Illinois Institutional Animal Care and Use Committee. Male C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were housed in microisolator cages under specific pathogen-free conditions, fed with autoclaved food, and used in experiments at 8–12 wk of age (25–30 g).
Depletion of calpain 1 and calpain 2 in mouse lungs.
Calpain 1 and 2 small interfering RNAs (siRNAs) are a pool of three target-specific 20–25 nt siRNAs (Santa Cruz Biotechnology, Santa Cruz, CA). Calpain 1/2 was depleted in mouse pulmonary vasculature by retro-orbital injection of cationic liposome-siRNA complexes as described previously (16, 41). The liposome-siRNA complexes were prepared by addition of 0.5 mg/kg siRNA into 150 μl of liposome suspensions. A scramble siRNA was employed as control. Successful transfection of calpain 1 and calpain 2 siRNAs was confirmed by Western blotting analysis of lung homogenates. All animal experiments were performed 48 h after transfection.
We used a well-established in vivo mouse model of VILI as described previously (15, 22). Briefly, mice were anesthetized with ketamine (75 mg/kg), underwent tracheotomy, and were ventilated for 2 h with 1% isoflurane in room air to maintain anesthesia, using either high or normal tidal volume. For high tidal volume ventilation, mice were connected to a Harvard Apparatus ventilator (MiniVent; Harvard Biosciences, Holliston, MA), with a tidal volume of 28 ml/kg, a respiratory rate of 60 breaths/min, and 0 cmH2O end-expiratory pressure, For normal volume ventilation, mice received 7 ml/kg of tidal volume, a respiratory rate of 120 breaths/min, and 0 cmH2O end expiratory pressure. To maintain PCO2 between 35 and 45 Torr (4.7–6.0 kPa), ∼5 ml of dead space was added to the ventilator circuit. Body temperature was maintained between 37°C and 38°C, using a heating lamp (15). In some experiments, animals were pretreated with inhibitor of both calpain 1 and calpain 2, calpain inhibitor I (ALLN, 5–20 mg/kg ip), NOS inhibitor nitro-l-arginine methyl ester (l-NAME) (15 mg/kg ip) or tyrosine phosphorylation inhibitor genistein (50 mg/kg ip) 1 h before exposure to mechanical ventilation. After 2 h of mechanical ventilation, various measurements were obtained.
Calpain activity assay.
Analysis of calpain activity in the lung was performed using a calpain activity assay kit (Abcam, Cambridge, MA) according to the manufacturer's instructions. Briefly, lungs were homogenized in lysis buffer at 4°C. Clarified cell lysates were then incubated with substrate (Ac-LLY-AFC) and reaction buffer for 1 h at 37°C in the dark. Upon cleavage of substrate, the fluorogenic portion (7-amino-4-trifluoromethyl coumarin) releases yellow-green fluorescence at a wavelength of 505 nm following excitation at 400 nm. Fluorescence emission was measured by a standard fluorimeter. For each sample, control reactions were performed in the presence of 5 μg of recombinant calpastatin (Calbiochem-Novabiochem, San Diego, CA) to monitor any calpain-independent proteolysis of fluorogenic peptide. Values from control reactions were subtracted from total activity values to specifically determine calpain activity for each sample. Results are expressed as relative fluorescence units per milligram of lysate protein.
Western blot analysis.
At the end of experiments, lungs were homogenized and lysed in lysis buffer (50 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1.0% NP-40, 0.1% SDS, 1 mM Na3VO4, 1 mM NaF, 1 mM PMSF, and protease inhibitor mixture). Protein concentrations were determined by the bicinchoninic acid (BCA) assay. Equal amounts of protein from each lysate were separated by SDS/PAGE and transferred to nitrocellulose membranes, blocked, and then incubated with relevant blotting antibodies. Protein bands were visualized using Pierce ECL reagent. Densitometric measurement was preformed from scanned films using ImageJ software (NIH) (16, 41).
NO production measurement.
NO production in the lung was determined using a commercial nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, lung samples were homogenized, filtered, and centrifuged. The supernatant (100 μl) was mixed with 100 μl of Griess reagent, and after a 10-min incubation at room temperature, the absorbance was measured at 540 nm with a 96-well plate reader. Nitrite concentration was calculated using sodium nitrite as a standard.
Lung MPO assay.
The azurophilic granules of PMNs contain MPO, a potent oxidizing and chlorinating agent, which can be used as a marker for PMN infiltration into the lung (15, 41). MPO activity was determined using a MPO assay kit (Invitrogen) according to manufacturer's protocol. Briefly, at the end of experiment, lungs were immediately removed, frozen, and stored at −80°C until assayed. Lung tissue (∼50 μg) was homogenized in 1 ml of cold sample buffer, sonicated three times for 15 s on ice, and centrifuged at 16,000 g for 30 min at 4°C. Protein concentrations were determined by BCA protein assay. MPO activity was determined in 100 μl of supernatants in duplicate using development reagent. Activity was measured over 25 s at 450 nm. Development reagent without sample was used as a control. MPO activity was expressed as change in absorbance per milligram of protein.
Determination of PMN counts in bronchoalveolar lavage fluid.
At the end of experiments, bronchoalveolar lavage (BAL) was performed by intratracheal injection of 1 ml of PBS followed by gentle aspiration. The lavage was repeated three times. The pooled BAL fluid was centrifuged at 400 g for 5 min, and cell pellets were suspended in PBS. Cell suspensions were diluted to a final concentration of 1 × 105 cells/ml, and a 200-μl volume of resuspended cells was cytospun onto slides at 300 revolutions/min for 5 min with a cytocentrifuge (Shandon, Southern Sewickley, PA). Slides were stained with Diff-Quick dye (Dade Behring, Newark, DE) and examined at a magnification of ×20 and ×40 by light microscopy. The percentage of PMNs was determined after counting 300 cells in randomly selected fields. The total cell count in BAL fluid was manually measured using a hemocytometer.
Assessment of pulmonary vascular permeability and edema formation.
Protein concentration in BAL was determined with BCA method as an index of increased permeability of the alveolar-capillary barriers (15, 41). Extravascular lung water (ELW) was used as an index of lung water content and edema (20). At the end of experiments, the lungs were removed, weighed, and homogenized after addition of 1 ml of distilled water. The lung homogenate was weighed, and an aliquot was centrifuged (16,000 g, 10 min) for assay of hemoglobin concentration in the supernatant. Blood was collected through right ventricular puncture. A second aliquot of homogenate, supernatant, and blood was weighed and then desiccated in an oven (60°C for 48 h) for gravimetric determination of ELW. The wet-to-dry lung weight ratio (lung W/D ratio) was calculated by a standard formula. The following formula was used to calculate ELW: (lung W/D ratioexperimental × lung dry weightexperimental) − (lung W/D rationormal × lung dry weightexperimental) × 1,000.
Levels of TNF-α and IL-6 in BAL were measured using commercial ELISA kits (R&D Systems, Minneapolis, MN and BD Biosciences, San Jose, CA) according to the manufacturer's instructions (11). Each value represents the means of triplicate determinations.
Drugs, reagents, and antibodies.
The chemicals and reagents used were obtained from Sigma Chemical (St. Louis, MO) unless otherwise stated. l-NAME (Cayman Chemical), calpain inhibitor I, and genistein were dissolved in DMSO. Calpain 1 MAb was from Abcam. NOS-3 and phospho-NOS-3 polyclonal antibodies were from Millipore (Billerica, MA); ICAM-1, phospho-ICAM-1, calpain 2, inducible NOS (NOS-2) polyclonal antibodies, and horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG antibodies from Santa Cruz Biotechnology. Akt and phospho-Akt MAbs were from Cell Signaling (Danvers, MA).
One-way analysis of variance and Student's Newman-Keuls test for post hoc comparisons were used to determine differences between control and experimental groups. Student's t-test was performed for paired samples. Parameter changes between different groups over time were evaluated by a two-way analysis of variance with repeated measures. Data were expressed as means ± SE. A P value <0.05 was considered significant.
Rapid calpain activation by high tidal volume mechanical ventilation in the mouse lung.
To elucidate the role of calpain in the regulation of ventilator-induced lung inflammation, we first determined whether mechanical ventilation causes calpain activation in the lung. We observed that calpain activity in the lung doubled within 15 min after mechanical ventilation with high tidal volume (28 ml/kg) and maintained a maximal level for at least 2 h (Fig. 1A). Calpain activity was not altered within 2 h of normal ventilation (7 ml/kg). Calpain inhibitor I reduced calpain activity in a concentration-dependent manner, with a complete inhibition of mechanical ventilation-induced calpain activation at the concentration of 10 mg/kg and below the basal level of calpain activity at the concentration of 20 mg/kg, respectively (Fig. 1B). Accordingly, 10 mg/kg of calpain inhibitor I was chosen and used in the subsequent experiments.
Calpain inhibitor attenuates mechanical ventilation-induced lung inflammation.
In view of mechanical ventilation-induced calpain activation, we next evaluated the effect of a selective calpain inhibitor in VILI. Accordingly, mice were treated either with the calpain inhibitor I or with vehicle (DMSO). No mice showed any signs of toxicity caused by the drug or the vehicle, and all survived the ventilatory period. The effects of calpain inhibitor on the integrity of the alveolar-capillary barrier and pulmonary edema formation were examined by measuring the concentration of total protein in BAL fluid and ELW, respectively. Mechanical ventilation with higher tidal volume induced an increase in transalveolar protein permeability and lung edema formation compared with normal ventilation, consistent with our previous findings (15) and those of others (22). Mice treated with calpain inhibitor I showed lower protein permeability and less lung edema than vehicle-treated mice after 2 h of high tidal volume ventilation (Fig. 2, A and B). As PMN adhesion to pulmonary vascular endothelial cells and migration into the alveolar space are critical for induction of lung inflammation (1), we investigated the role of calpain in the mechanism of PMN infiltration into the lung. As expected, mechanical ventilation with high tidal volume caused a marked increase in PMN counts in BAL fluid in vehicle-treated mice (Fig. 2C) and lung MPO level (Fig. 2D). PMN infiltration into the lung was significantly reduced in mice pretreated with calpain inhibitor I (Fig. 2, C and D). Furthermore, treatment with calpain inhibitor I was associated with a decrease in TNF-α and IL-6 in BAL fluid (Fig. 2, E and F). Calpain inhibitor had no effect on pulmonary vascular permeability, edema formation, PMN infiltration, and production of TNF-α and IL-6 in mice ventilated with normal tidal volume. Collectively, these results demonstrate that selective pharmacological inhibition of calpain activation has a positive effect on VILI in this experimental model.
Depletion of calpain 1 or calpain 2 in pulmonary vasculature reduced mechanical ventilation-induced lung inflammation.
To determine the relative contributions of two calpain isoforms, calpain 1 and calpain 2, to VILI, we separately knocked down either calpain 1 or calpain 2 with specific siRNAs in pulmonary vasculature in vivo using liposome-siRNA complex technique (16, 41). Lung transfected with calpain 1 siRNA exhibited decreases in calpain 1 protein levels (Fig. 3A), but levels of calpain 2 in lung homogenates were similar to those of nonsilenced control lungs (Fig. 3A). Conversely, lung transfected with a specific calpain 2 siRNA showed calpain 1 levels similar to control, whereas calpain 2 protein levels in these lungs were significantly reduced (Fig. 3B). Quantification of calpain 1 and calpain 2 by Western blot analysis revealed a greater than 80% reduction in respective target protein levels in the knockdown mouse lungs (Fig. 3, A and B). Consistently, calpain activity was significantly reduced by ∼85% in calpain 1 or calpain 2 siRNA-treated lungs compared with scramble siRNA-transfected lungs following mechanical ventilation. Mechanical ventilation per se did not alter calpain 1 and calpain 2 protein expressions in the lung.
Mechanical ventilation with high tidal volume induced an increase in transalveolar protein permeability and lung edema formation in mice treated with scrambled siRNA. In agreement with the inhibition of calpain activity, depletion of calpain 1 or calpain 2 in pulmonary vasculature attenuated the vascular leakage and lung edema in VILI, as indicated by the significant decrease in total concentrations of BAL protein and ELW after exposure to mechanical stress associated with high tidal volume mechanical ventilation (Fig. 3, C and D). PMN counts in BAL fluid (Fig. 3E) and lung MPO level (Fig. 3F) also increased in the mechanically ventilated, scrambled siRNA-treated lungs. These responses were significantly abolished in mice transfected with calpain 1 or calpain 2 siRNA (Fig. 3, E and F). Furthermore, calpain 1 or calpain 2 knockdown inhibited mechanical ventilation-induced increases in BAL concentrations of TNF-α and IL-6 (Fig. 3, G and H). Calpain 1 or calpain 2 knockdown had no effect on pulmonary vascular permeability, edema formation, PMN infiltration, and production of TNF-α and IL-6 in mice ventilated with normal tidal volume (data not shown). These results suggest that calpain 1 and calpain 2 act cooperatively and play important roles in mediating ventilator-induced lung inflammation and injury.
Calpain activation regulates ICAM-1 phosphorylation during VILI.
Tyrosine phosphorylation of ICAM-1 has recently been shown to mediate leukocyte transmigration during the early stages of inflammation (34, 36). We thus tested the hypothesis that mechanical ventilation with high tidal volume may induce ICAM-1 phosphorylation via calpain activation. ICAM-1 phosphorylation at Tyr518 in the lung tissue started to increase at 15 min after mechanical ventilation and lasted for at least 2 h (Fig. 4A). Pretreatment of mice with calpain inhibitor I significantly attenuated mechanical ventilation-induced ICAM-1 tyrosine phosphorylation (Fig. 4B). Similarly, mechanical ventilation caused an increase in ICAM-1 phosphorylation at Tyr518 in mouse lungs treated with a scrambled siRNA. Depletion of calpain 1 or calpain 2 in the pulmonary vasculature with a specific siRNA prevented mechanical ventilation-induced ICAM-1 phosphorylation (Fig. 4C). Calpain inhibitor and calpain 1 or calpain 2 siRNA had no effect on basal ICAM-1 phosphorylation. Notably, 2-h mechanical ventilation with high tidal volume (28 mg/kg) did not alter ICAM-1 protein expression (Fig. 4A), consistent with the previous findings (13). These findings suggest that calpain mediates ICAM-1 tyrosine phosphorylation during VILI.
Calpain-mediated NO production stimulates ICAM-1 phosphorylation and subsequent PMN infiltration into the lung.
NO generated from both NOS-3 and NOS-2 is a crucial in vivo determinant of lung inflammation (10), and NO has been implicated in the pathogenesis of VILI (42). We therefore examined the role of calpain in the regulation of NO product during mechanical ventilation. Time-course experiments revealed that NO concentration (measured as nitrite metabolite) in the lung was abruptly increased threefold 15 min after mechanical ventilation and then slowly increased within 2 h (Fig. 5A). Two-hour ventilation caused an approximately fourfold increase in NO concentration in the lung, whereas calpain inhibitor prevented this effect (Fig. 5B). Calpain inhibitor did not alter the basal NO concentration in the lung. Similarly, mechanical ventilation significantly increased NO concentration in the lung in mice treated with a scrambled siRNA. Depletion of calpain 1 or calpain 2 inhibited ventilation-induced increase in NO concentration in the lung (Fig. 5C). There was no significant difference in basal concentration of NO in the lung in all groups (data not shown).
NO has been shown to stimulate tyrosine phosphorylation of a set of proteins (30, 33). In the present study, we focused on identifying whether mechanical ventilation with high tidal volume induced ICAM-1 phosphorylation and subsequent PMN migration through NO-dependent mechanism in mouse lung. As shown in Fig. 6, nonspecific NOS inhibitor l-NAME abolished ICAM-1 phosphorylation at Tyr518 (Fig. 6A) and significantly attenuated the number of PMN in BAL (Fig. 6C) during VILI. To verify the effect of ICAM-1 tyrosine phosphorylation on PMN infiltration into the lung, we pretreated the mice with genistein, a specific inhibitor of protein tyrosine kinase. Genistein pretreatment completely abolished ICAM-1 phosphorylation at Tyr518 (Fig. 6B) and markedly decreased PMN infiltration into the lung after exposure to high tidal volume mechanical ventilation. l-NAME also attenuated the basal level of ICAM-1 phosphorylation (Fig. 6A). l-NAME and genistein had no effect on PMN count in BAL in mice ventilated with normal tidal volume (Fig. 6C). There was no change in ICAM-1 protein expression in all groups.
A critical role of calpain in NOS-3-derived NO production after mechanical ventilation.
We observed that mechanical ventilation with high tidal volume induced NO product in the lung through both NOS-3 and NOS-2 pathways as evidenced by increased NOS-3 phosphorylation (activation) and NOS-2 protein expression (Fig. 7A). NOS-3 phosphorylation increased by 2.5-fold 15 min after ventilation, remained high level the next 45 min and then decreased 2 h after ventilation (Fig. 7A). NOS-3 protein levels were similar and remained constant at all time points following mechanical ventilation (Fig. 7A). Calpain inhibitor I significantly reduced NOS-3 phosphorylation but not NOS-2 protein expression during VILI (Fig. 7B). Calpain inhibitor I did not alter basal levels of NOS-3 phosphorylation and NOS-2 expression. Similarly, depletion of calpain 1 or calpain 2 with a specific siRNA attenuated NOS-3 phosphorylation but not NOS-2 expression (Fig. 7C). These results suggest that calpain mediates NO production via NOS-3-dependent pathway. To confirm the role of calpain in NOS-3 activation, we next determined the effect of calpain on activation of Akt, an upstream signaling molecule of NOS-3. Following high tidal volume mechanical ventilation, the phosphorylation of Akt was increased in a time-dependent manner within 60 min, followed by a decrease at 120 min. There was no significant change in the expression of total proteins of Akt during the whole ventilatory period (Fig. 7A). Calpain inhibitor prevented mechanical ventilation-induced Akt phosphorylation (Fig. 7B). Similarly, depletion of calpain 1 or calpain 2 with specific siRNAs attenuated Akt phosphorylation in response to high tidal volume ventilation (Fig. 7C). Calpain inhibitor and calpain siRNAs did not alter levels of Akt phosphorylation in mice ventilated with normal tidal volume.
Lung inflammatory responses attributable to mechanical stretch in the absence of infection have been considered as one of major mechanisms of VILI (11). It is therefore medically important to understand the basis and molecular mechanisms of this “sterile inflammation” in the lung provoked by physical forces. Mechanical forces should be initially sensed and then converted into the biochemical signaling that stimulates inflammatory cell infiltration into the lung and release of proinflammatory cytokines, resulting in lung injury. Accordingly, better understanding of the early signaling of mechanotransduction may help design specific treatment strategies that block the susceptibility of lung cells to mechanical forces, which may reduce VILI and improve outcome in patients. In the present study, we identify calpain activation as an early event of lung inflammation in response to high tidal volume mechanical ventilation. Activity assay showed that calpain in the lung was rapidly activated (within minutes) following mechanical ventilation. Either inhibition of calpain activity by a pharmacological agent or genetic disruption of calpain expression by gene silencing substantially attenuated ventilator-induced lung inflammatory injury. These findings strongly suggest that improper activation of calpain due to mechanical stretch mediates early lung inflammation.
The enzymatic activity of calpains depends on the Ca2+ concentration present inside the cell. The enzymatically active fragment of calpains contains two Ca2+-binding sites within the catalytic core. Ca2+ binding to these sites is cooperative and required for full enzymatic activity (12, 29). Accumulating evidence indicates that mechanical stretch induces an increase in the intracellular Ca2+ concentration in pulmonary microvascular endothelial cells (19), alveolar epithelial cells (43) and isolated lung preparation (31). It therefore seems likely that mechanical ventilation activates calpains via stretch-mediated elevation of intracellular Ca2+ concentration in our study. Although we did not determine the activity of calpains separately, the data from individual siRNA study demonstrated that both calpain 1 and calpain 2 are activated at an early stage of mechanical ventilation.
Calpain 1 and calpain 2 have been shown to be differentially involved in the regulation of inflammation (17). Therefore, we attempted to determine the specific role of each calpain isoform in the development of lung inflammation in response to mechanical ventilation. Surprisingly, we found that depletion of calpain 1 or calpain 2 in mouse pulmonary vasculature in vivo had a protective effect against ventilator-induced lung inflammation and injury. Consistent with the finding that calpain 1 and calpain 2 in platelets have similar physiological functions and pathological actions (5, 21), our results indicate that both calpain 1 and calpain 2 in the lung contribute to ventilator-induced lung inflammation and injury.
PMN migration into lung interstitium and the alveolar space is critical for induction of lung inflammatory injury (1, 15, 41). We observed that inhibition of calpain activity using calpain inhibitor I or calpain siRNAs decreased PMN sequestration in the lung, suggesting an important role of calpain in positively regulating lung PMN sequestration induced by mechanical stretch. Tyrosine phosphorylation of ICAM-1 has been reported to regulate ICAM-1 function and promote early leukocyte transmigration (34, 36). Recently, our study demonstrated that phosphorylation of ICAM-1 at Tyr518 induced a rapid PMN adhesion to mouse pulmonary vascular endothelium 5 min after TNF-α stimulation. Exogenous expression of a nonphosphorylatable (Tyr518Phe) ICAM-1 mutant mouse cDNA in ICAM-1−/− mouse lungs significantly blocked PMN infiltration into the lung after lipopolysaccharide challenge (24). The present findings extend these observations by indicating that mechanical ventilation with high tidal volume caused a time-dependent ICAM-1 phosphorylation at Tyr518 and subsequent PMN infiltration into the lung. Furthermore, selective inhibition of tyrosine phosphorylation of ICAM-1 abolished mechanical stretch-induced PMN infiltration into the lung. Increased ICAM-1 protein expression has been shown to play a major role in the recruitment of PMNs in lungs during long-term mechanical ventilation (23, 28). In the present study, however, we observed no alteration in ICAM-1 protein expression following short-term mechanical ventilation (2 h), consistent with previous observations (13). Thus it seems unlikely that enhanced ICAM-1 protein expression is involved in ventilation-induced PMN infiltration into the lung in our experimental model. Importantly, inhibition of calpain by calpain inhibitor or by siRNA directed against calpain 1 or calpain 2 did not alter ICAM-1 protein expression but blocked ICAM-1 phosphorylation at Tyr518 and PMN migration caused by high tidal volume ventilation. Taken together, these findings support the novel concept that calpain activation mediates the early neutrophilic lung inflammation during mechanical ventilation via modulation of phosphorylation of ICAM-1.
The molecular mechanisms by which calpain activation regulates the state of ICAM-1 phosphorylation at Tyr518 during mechanical ventilation remain unknown. In a mouse model of acute lung inflammation, Src kinase has been demonstrated to be able to phosphorylate ICAM-1 following TNF-α treatment (24). In the present study, nonspecific NOS inhibitor l-NAME prevented mechanical stretch-induced ICAM-1 phosphorylation and subsequent PMN infiltration into the lung, supporting the possibility that NO may be a stimulator of ICAM-1 phosphorylation. NO reacts with superoxide anion to form peroxynitrite. Peroxynitrite not only can nitrate tyrosine residues of many proteins (6, 18, 37) but also can irreversibly inactivate phosphotyrosine phosphatases that might result in an increase in tyrosine phosphorylation (26, 39). NO has also been shown to activate Src kinases (30). Our results revealed that calpain inhibition by means of genetic and pharmacological approaches significantly reduced mechanical ventilation-induced NO product and ICAM-1 phosphorylation, suggesting that calpain activation during mechanical ventilation stimulates NO release, which in turn results in ICAM-1 phosphorylation.
It has been previously established that mechanical ventilation with high tidal volume stimulates NO production in the lung although its molecular mechanisms are still controversial (42). Mechanical ventilation induced NOS-2 protein expression in rat lung (9), and specific inhibition of NOS-2 attenuates VILI (32). Mice deficient in NOS-2 were also protected from pulmonary inflammation and injury in response to mechanical ventilation with high tidal volume (32). In other studies, VILI was markedly attenuated in NOS-3-deficient mice (35, 40). In the present study, we observed that a specific calpain inhibitor and siRNA against calpain 1 or calpain 2 blocked Akt/NOS-3 activation, but not NOS-2 expression, suggesting a role of NOS-3/NO pathway in calpain signaling lung inflammation induced by mechanical ventilation with high tidal volume. The role of calpain in the regulation of NOS-3/NO signaling remains controversial. In the presence of calcium, purified calpain enzymes were found effective in cleaving NOS-3 that is pulled down from endothelial cell lysates using a monoclonal antibody for HSP90 (4). Activation of protease calpain by oxidized and glycated LDL increases the degradation of NOS-3 (9). In porcine pulmonary arterial endothelial cells, specific calpain inhibitors prevented the hypoxia-induced loss of NOS-3 activity (38). However, in bovine aortic endothelial cells, calpain mediates AMPK-dependent NOS-3 activation in response to VEGF via its interaction with ezrin (44). Our findings demonstrated that calpain activation attributable to mechanical ventilation activated Akt/NOS-3 signaling pathway. Although detailed mechanisms underlying calpain activation of Akt remain to be fully investigated, our data no doubt have unraveled an important role of calpain-mediated Akt-NOS-3/NO signaling at an early stage of lung inflammation induced by mechanical ventilation.
In summary, our findings provide evidence supporting the idea that calpain mediates ventilator-induced lung inflammation and injury. Mechanical stretch causes a rapid activation of calpain, which upregulates NOS-3 activity via an Akt-dependent pathway. Excessive NO generated by NOS-3 and NOS-2 induces massive lung inflammation through Src-mediated ICAM-1 phosphorylation and subsequent PMN infiltration, ultimately resulting in lung injury (Fig. 8). Thus our study identifies calpain activation as an early event of lung inflammatory injury induced by hyperinflation. These findings suggest that calpain may represent a novel therapeutic target for the prevention and treatment of VILI.
This work was supported by NIH NHLBI grant 5R01HL104092 (G. Hu) and American Heart Association Scientist Development Grant 0730331N (G. Hu).
The authors have no financial conflict of interest.
We thank Maricela Castellon (Depts. of Pharmacology and Anesthesiology, University of Illinois College of Medicine) for technical assistance.
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