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Am J Physiol Lung Cell Mol Physiol 292: L1327-L1334, 2007. First published February 16, 2007; doi:10.1152/ajplung.00380.2006
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TRANSLATIONAL PHYSIOLOGY

MLCK210 gene knockout or kinase inhibition preserves lung function following endotoxin-induced lung injury in mice

Janet L. Rossi,1,2 Anastasia V. Velentza,1,3 David M. Steinhorn,2 D. Martin Watterson,1,3 and Mark S. Wainwright1,2,3

1Center for Drug Discovery and Chemical Biology and Departments of 2Pediatrics and 3Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, Illinois

Submitted 26 September 2006 ; accepted in final form 13 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Barrier dysfunction, involving the endothelium or epithelium, is implicated in the pathophysiology of many disease states, including acute and ventilator-associated lung injury. Evidence from cell culture, in vivo and clinical studies, has identified myosin light chain kinase as a drug discovery target for such diseases. Here, we measured disease-relevant end points to test the hypothesis that inhibition of myosin light chain kinase is a potential therapeutic target for treatment of barrier dysfunction resulting from acute lung injury. We used a combined gene knockout and chemical biology approach with an in vivo intact lung injury model. We showed that inhibition of myosin light chain kinase protects lung function, preserves oxygenation, prevents acidosis, and enhances survival after endotoxin exposure with subsequent mechanical ventilation. This protective effect provided by the small molecule inhibitor of myosin light chain kinase is present when the inhibitor is administered during a clinically relevant injury paradigm after endotoxin exposure. Treatment with inhibitor confers additional protection against acute lung injury to that provided by a standard protective mode of ventilation. These results support the hypothesis that myosin light chain kinase is a potential therapeutic target for acute lung injury and provide clinical end points of arterial blood gases and pulmonary compliance that facilitate the direct extrapolation of these studies to measures used in critical care medicine.

endothelium; acute respiratory distress syndrome; protein kinase; oxygenation

TISSUE BARRIER DYSFUNCTION resulting from endothelial and or epithelial dysfunction (6, 12, 20, 29, 33) is a common pathophysiology among multiple diseases and may also occur as an iatrogenic complication of therapies. Epithelial dysfunction occurs in diseases such as inflammatory bowel disease (8) and as a result of graft vs. host disease following stem cell transplantation. Endothelial dysfunction occurs in disease states such as acute lung injury (ALI) (3, 14, 32), burns (27), sepsis, and multiorgan dysfunction syndrome (12, 33). The morbidity suffered by patients with barrier dysfunction as a result of disease or therapies has brought about a renewed interest in the development of treatments targeting barrier dysfunction.

Current hypotheses identify dysfunction of the endothelial cell (EC) layer as a pivotal event in the pathophysiology of barrier dysfunction (28). The discovery of therapeutic targets associated with EC dysfunction has the attraction that efficacy in one disease area could have potential broad utility across multiple disease areas in critical care medicine (2, 7, 9, 16). The endothelial form of the Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK) MLCK210 plays a critical role in the control of EC barrier function (28). Phosphorylation of myosin light chain (MLC) is a pivotal regulatory step leading to contraction of the cellular cytoskeleton and an increase in EC paracellular gap formation (26). Cell-based and in vivo studies have demonstrated impairment of EC barrier function associated with increase in MLCK activity and changes in cytoskeletal structure (13, 22). We previously reported (31) that MLCK (MLCK210) knockout (KO) mice are protected against endotoxin-induced ALI and the lethal complications of a subsequent ventilator-associated lung injury (VALI). We observed comparable protection against lung injury in wild-type (WT) mice pretreated with a novel, bioavailable inhibitor of MLCK. The convergence of data from the in vivo gene KO model and the pharmacological inhibitor study identified endothelial MLCK (MLCK210) as a potential therapeutic target in the treatment of ALI and VALI.

To establish a foundation for the translation of these studies into clinical practice, we used MLCK210 KO mice and a small molecule inhibitor of MLCK in a disease-relevant mouse model of ALI and VALI. To determine the role of MLCK210 in the compromise of lung function in ALI and VALI, and to link these findings to end points used in a clinical practice, we measured changes in lung compliance, arterial blood gases (ABGs), and pulmonary edema in response to endotoxin-induced ALI and ALI combined with VALI. To determine more directly the potential of MLCK210 as a therapeutic target in clinical practice, we tested the MLCK inhibitor in a chemotherapeutic mode after exposure to endotoxin and measured changes in lung compliance and gas exchange. These results support the role of MLCK210 as a key endothelial barrier regulatory molecule in the pathophysiology of lung injury, establish an in vivo link between EC and lung function, and add further support to the validation of MLCK as a therapeutic target in the management of lung injury.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Adult (20–30 g) C57Bl/6 mice of both genders were obtained from a commercial vendor (Harlan, Indianapolis, IN). The generation and characterization of the MLCK210 KO mouse strain was previously described (31). All animal procedures were performed in accordance with relevant National Institutes of Health guidelines and approved by the Institutional Animal Care and Use Committee of Northwestern University.

Treatment groups. Each group consisted of six mice (n = 6) unless otherwise specified. The treatment groups consisted of 1) KO saline controls, 2) KO treated with LPS (Salmonella typhimurium; Sigma, St. Louis, MO), 3) WT saline controls, 4) WT treated with LPS, and 5) WT treated with LPS followed by MLCK inhibitor.

Injections of mice. All groups received equally timed injections based on randomized group assignment. All compounds, LPS, inhibitor, or an equal volume of sterile saline diluent were administered via intraperitoneal (ip) injection. LPS was administered at a dose of 10 mg/kg body wt as previously described (31). Control mice received an equivalent volume of diluent. Inhibitor was injected at a dose of 2.5 mg/kg 4 and 24 h after LPS injection. Other groups were injected with an equal volume of diluent.

MLCK inhibitor. The synthesis and characterization of the MLCK inhibitor were as previously described (18, 31). In brief, the approach used to discover 11-(3-chloro-6H-pyridazin-1yl)-undecanoic acid (6-phenyl-pyridazin-3-yl)-amide was used previously for protein kinase inhibitors on the basis of the privileged 3-aminopyridazine scaffold. The reactions were monitored by analytical HPLC, and the final product was purified by reverse-phase HPLC. Kinase inhibitory activity was tested against death-associated protein kinase, calmodulin-regulated protein kinase II, GSK3B, and Rho kinase. The concentrations of the inhibitor, up to 100 µM, that failed to inhibit these relevant kinases are >20 times the Ki value of 5 µM, supporting the selectivity of the inhibitor for MLCK.

Ventilation of mice. Twenty-four hours after LPS injection, mice from each treatment group were ventilated with one of two protocols. To determine the role of MLCK in survival after ALI combined with VALI, mice were ventilated with an aggressive ventilator protocol designed to result in VALI (21). These mice were endotracheally intubated and ventilated under isoflurane anesthesia for 60 min with large tidal volumes (10–12 ml/kg), no positive end-expiratory pressure (PEEP), 100% FIO2, and a rate of 180 breaths/min (HSE MiniVent; Harvard Apparatus, Holliston, MA). To determine the role of MLCK210 in changes in compliance associated with VALI, mice were ventilated using a protective ventilator protocol (21) commonly used in clinical practice. These mice were intubated and ventilated for 2 h with low tidal volumes (6–8 ml/kg), PEEP of 3 cmH2O pressure, 100% fractional inspired oxygen (FIO2), and a rate of 180 breaths/min. At the conclusion of ventilation, mice were killed by pentobarbital overdose. Static pressure-volume (compliance) measurements were performed, ABGs were obtained, and lungs were removed for light microscopic analysis. Core temperature was monitored during ventilation using a rectal probe (Physitemp, IT-18), and normothermia (37.0 ± 0.1°C) was maintained with servo-controlled surface heating and cooling.

Survival end point assays. All mice (LPS exposure and MLCK inhibitor treatment as described above) (MLCK KO; n = 3) were subjected to the aggressive ventilator protocol with the same parameters for 60 min, and survival was monitored and recorded.

Compliance measurements. Compliance measurements were obtained on mice using the treatment groups described above and the protective ventilator protocol. Compliance measurements were obtained from ventilated and control (nonventilated) mice at the conclusion of the 24-h treatment period. Pressure measurements were obtained after 2 h of ventilation using a Pro-paq 106 monitor, a portable monitor that measures blood pressure (Protocol Systems), and a pressure transducer calibrated according to the manufacturer's recommendations. Air was introduced into the endotracheal tube in 50-µl increments utilizing a calibrated syringe to a total volume of 750 µl. Pressure was recorded from the Pro-paq monitor, and volume was corrected for the weight of the mouse to generate a static compliance curve. Individual pressure points were obtained per unit of volume in duplicate. Each point for each mouse in a group was then averaged, and the average ± SE was plotted per volume point to form the curve. The curve was then plotted, and the slope was obtained from that curve using a nonlinear regression approach with the F-test (GraphPad Prism version 4). The slope was obtained from the part of the curve between the low and high inflection points. Conversion was made from mmHg to cmH2O using a factor of 1.36.

ABGs. At the conclusion of the 24-h treatment period, all blood gas measurements were obtained on mice (LPS exposure and MLCK inhibitor treatment described above) that were subjected to the protective mode of ventilation for 2 h. Arterial blood was drawn from the left ventricle at the conclusion of ventilation and measured on a clinical I-STAT (Abbott Labs, Chicago, IL) monitor with GCS +8 cartridges. The monitor and cartridges were calibrated and used according to the manufacturer's recommendations.

Gravimetric assessment of pulmonary edema. Four hours following LPS injection, WT mice received either inhibitor (2.5 mg/kg) or equal volume of diluent and were killed 20 h later. These mice did not undergo ventilation. Lungs were removed and weighed using a high-precision balance (Mettler Instruments; Mettler-Toledo, Columbus, OH). The lungs were dried at 60°C for 48 h before reweighing. The wet weight was divided by the dry weight, and the ratio was compared between groups (23).

Histological assessment of lung injury. Lungs were harvested from WT and KO mice exposed to LPS, or LPS combined with MLCK inhibitor, using the same treatment regimen described for measurements of changes in compliance. All mice underwent 2 h of protective mode of ventilation before they were killed. Following perfusion and fixation in formalin, paraffin-embedded hematoxylin and eosin (H&E)-stained sections were prepared by standard techniques. Assessment of injury was performed in blinded fashion by grading four histological findings: hemorrhage; inflammation; atelectasis; and edema, as previously described (31).

Dose dependence of MLCK inhibitor treatment. To determine dose dependence of the response to treatment with the MLCK inhibition, endotoxin-treated WT mice received one of four doses of MLCK inhibitor: 0.25 mg/kg, 0.75 mg/kg, 1.5 mg/kg, or 2.5 mg/kg 4 h after LPS injection. Twenty-four hours after LPS exposure, mice underwent the lung protective mode of ventilation. Static compliance was measured after 2 h of ventilation.

Statistical analysis. The slopes of compliance curves calculated from the end of the inflation curves were compared between groups by nonlinear regression methods using extra sum-of-squares F-test (GraphPad Prism version 4.00, GraphPad Software). Differences in Kaplan-Meier survival curves were calculated by Mantel-Haenszel log rank test. Wet/dry weights were analyzed with Mann-Whitney nonparametric Student's t-test. ABGs were analyzed with ANOVA with appropriate corrections for multiple comparisons. Data are presented as means ± SE except lung injury scores, which are expressed as median ± interquartile range. Statistical significance was assumed when P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effects of MLCK210 gene KO or MLCK inhibitor on survival following combined ALI and VALI. We first evaluated the combination of endotoxin-induced ALI with VALI. We used a bioavailable small molecule MLCK inhibitor (31) to determine the role of MLCK in the increased mortality associated with combined ALI and iatrogenic VALI. We used a delayed treatment protocol in which the LPS-exposed WT mice were treated with the inhibitor 4 and 24 h following endotoxin exposure. WT mice were exposed to LPS for 24 h and then subjected to 60 min of an aggressive ventilator protocol to induce VALI (Fig. 1). All WT mice (n = 6) treated with LPS died within 20 min of ventilation. In contrast, WT mice treated with the MLCK inhibitor 4 and 24 h following endotoxin injection (n = 6) showed a significant improvement in survival (67%) when subjected to aggressive ventilation (P < 0.05 vs. WT+LPS). All saline-injected WT mice (n = 6), saline-injected KO mice (n = 3), and LPS-injected KO mice (n = 3) survived the 60-min ventilation period.


Figure 1
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  		Fig. 1. Inhibition of myosin light chain kinase (MLCK) by genetic knockout (KO) or small molecule inhibitor protects against LPS- and ventilator-induced death. Twenty-four hours after treatment with 10 mg/kg ip LPS, wild-type (WT) or KO mice were subjected to a 1-h period of aggressive ventilation in the absence of positive end-expiratory pressure (PEEP). The MLCK inhibitor (2.5 mg/kg ip) was administered to WT mice 4 and 24 h after LPS injection, just before initiation of ventilation. All LPS-injected WT mice (n = 6) died within 20 min after initiating ventilation (dashed lines). All control WT mice (n = 6), control KO mice (n = 3), and LPS-treated KO mice (n = 3) survived the 60-min test period of ventilation (dotted lines). There was a significant improvement in survival after 60 min of ventilation (67%) in endotoxin-exposed WT mice treated with the MLCK inhibitor (n = 6, solid line). WT+LPS vs. WT+LPS+inhibitor; P < 0.05 by Mantel-Haenszel log rank test. SAL, saline; INH, inhibitor.

 
Effect of MLCK210 gene KO or MLCK inhibitor on lung compliance following endotoxin exposure. Next, we compared the loss of lung compliance associated with endotoxin-induced ALI in WT and MLCK210 KO mice (Fig. 2). LPS-injected WT mice showed a significant reduction (0.776 ± 0.021; P < 0.05) in the slope of the lung compliance curve compared with control WT mice (0.885 ± 0.008) (Fig. 2A). The LPS- (1.202 ± 0.055) and saline-treated (1.280 ± 0.058) control MLCK210 KO mice showed indistinguishable compliance curves (Fig. 2B).


Figure 2
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  		Fig. 2. MLCK210 KO mice are protected from LPS-induced decrease in lung compliance. WT (bullet) or KO ({blacksquare}) mice were injected intraperitoneally with saline as controls or 10 mg/kg LPS (WT = {circ}, KO = {square}), and lung compliance was measured 24 h later. A: WT mice treated with LPS showed a significant decrease (P < 0.05) in the slope of the lung compliance curve compared with that of control WT mice. B: compliance curves obtained with control KO mice and LPS injected KO mice were not significantly different. Values are means ± SE. For some data points, the errors bars are not visible because they are smaller than the symbol; n = 6 for all groups.

 
Data from the previous experiments suggested that delayed treatment with the MLCK inhibitor could reduce mortality associated with combined ALI and severe VALI. To determine whether the same treatment regimen could attenuate the decrease in compliance associated with ALI and VALI, we combined endotoxin-induced lung injury with the protective ventilator strategy commonly used in clinical practice (7) and measured compliance under these conditions (Fig. 3). First, we confirmed that this ventilation protocol was protective. All LPS-injected WT mice survived 1 h of protective mode ventilation in contrast to the 100% mortality observed (Fig. 1) with the aggressive mode of ventilation. The slope of the compliance curve in WT mice following 2 h of ventilation after exposure to LPS was 0.714 ± 0.018, whereas WT mice treated with the MLCK inhibitor under the same conditions showed a significant (P < 0.05) improvement in compliance (0.980 ± 0.050), consistent with a protective effect of the MLCK inhibitor on lung function. Results using the MLCK inhibitor partially mimic the protection seen in KO mice exposed to this same combination of endotoxin and ventilation (1.250 ± 0.062).


Figure 3
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  		Fig. 3. Inhibition of MLCK by genetic KO or small molecule inhibitor improves lung compliance under conditions of protective ventilation using PEEP. Twenty-four hours after exposure to 10 mg/kg ip LPS, mice were subjected to ventilation using a protective ventilation strategy that included PEEP. After 2 h of ventilation, pulmonary compliance was measured. The slope of the compliance curve (n = 3) in LPS-exposed KO mice ({square}) was significantly higher (P < 0.05) than that of LPS-exposed WT mice (n = 5) ({triangleup}). The slope of the compliance curve in LPS-exposed WT mice treated with the MLCK inhibitor (n = 6) (bullet) was also significantly higher than that of WT+LPS mice (P < 0.05). Values are means ± SE. For some data points, the errors bars are not visible because they are smaller than the symbol.

 
Effect of MLCK210 gene KO or MLCK inhibitor on oxygenation and acid-base status following endotoxin exposure. To determine whether the preservation of lung compliance associated with MLCK210 gene KO or pharmacological inhibition of MLCK was associated with improved pulmonary gas exchange, we measured ABGs (n = 6) at the conclusion of the protective ventilation protocol (Fig. 4, A–D). Consistent with the preservation of lung compliance, MLCK gene KO or inhibitor treatment resulted in normalization of ABG values under these conditions. WT mice treated with LPS showed a significant reduction (P < 0.05) in pH (7.09 ± 0.04) and PaO2 (50.17 ± 1.64) compared with WT saline-treated control pH (7.38 ± 0.03) and PaO2 (287.5 ± 71.67). In contrast, WT mice exposed to LPS and then treated with inhibitor showed no significant acidosis (7.44 ± 0.04) or decrease in oxygenation (PaO2: 336.0 ± 42.16) and were not significantly different from non-endotoxin-exposed controls. MLCK210 KO mice exposed to endotoxin showed no significant difference in pH (7.37 ± 0.02) or PaO2 (343.2 ± 92.86) compared with either the WT saline control group or control KO mice treated with saline alone (7.32 ± 0.02 and 302.3 ± 78.36).


Figure 4
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  		Fig. 4. MLCK gene KO or treatment with MLCK inhibitor attenuates systemic acidosis (A) and preserves oxygenation (B) associated with endotoxin-induced lung injury. Arterial pH and PaO2 measured in control WT mice ({blacktriangleup}) administered saline and then ventilated for 2 h with the protective mode of ventilation was significantly different (P < 0.05) from pH measured in WT mice exposed to LPS (+) and ventilated under the same conditions. WT mice treated with LPS followed 4 h later with 2.5 mg/kg of inhibitor ({square}) showing protection from acidosis as well as preserved oxygenation (P < 0.05). KO mice showed no significant difference in pH or oxygenation between control ({blacklozenge}) and LPS treatment (dashes). PaCO2 (C) although significantly different for WT saline and LPS-exposed mice (P < 0.05), is not significantly different between remainder of groups. The hematocrit (D) is significantly higher (P < 0.05) in WT LPS-exposed mice compared with all groups. Values are means ± SE; n = 6 for all groups. *P < 0.05 by ANOVA.

 
The PaCO2 (40.87 ± 8.96) and the hematocrit (Hct) (46.17 ± 3.89) for the WT mice treated with LPS were significantly greater than in the WT saline controls (PaCO2: 20.6 ± 1.07 and Hct: 36.0 ± 0.89). Values for the KO saline controls (PaCO2: 28.6 ± 5.94 and Hct: 37.0 ± 1.39) were not significantly different from KO exposed to LPS (PaCO2: 28.3 ± 2.04 and Hct: 39.83 ± 3.07). Although WT mice exposed to LPS and treated with inhibitor showed no significant difference in PaCO2 (30.3 ± 4.07) or Hct (37.0 ± 1.095) compared with WT and KO controls and KO mice exposed to LPS, the PaCO2 (30.3 ± 4.07) was also not significantly different from WT mice exposed to LPS (40.87 ± 8.96).

Effect of MLCK210 gene KO or MLCK inhibitor on endotoxin-induced pulmonary edema. To determine whether protection afforded by the delayed administration of the MLCK inhibitor against endotoxin-induced lung injury was associated with preservation of EC barrier function, we measured lung wet/dry weights (Fig. 5) as an indicator of pulmonary edema after endotoxin-induced ALI (23). LPS-exposed WT mice were administered either saline or MLCK inhibitor at 2.5 mg/kg 4 h following endotoxin exposure, and lung weight measured after 24 h of endotoxin exposure. The wet/dry ratio in LPS-exposed WT mice treated with saline was 2.67 ± 0.05. The ratio in LPS-exposed mice treated with MLCK inhibitor (1.16 ± 0.03) was significantly reduced (P < 0.05) compared with mice treated with saline. The wet weight of the WT mice exposed to endotoxin and treated with saline (1.97 ± 0.02 g) was significantly increased (P < 0.5) compared with WT mice exposed to endotoxin and treated with MLCK inhibitor (0.79 ± 0.08 g).


Figure 5
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  		Fig. 5. Endotoxin-induced increase in lung edema in WT mice is prevented by delayed treatment with MLCK inhibitor. WT mice were injected with saline or 2.5 mg/kg inhibitor (n = 6/group) after 4 h of exposure to LPS (10 mg/kg). Gravimetric quantification of pulmonary edema was performed after 24 h of exposure to LPS. The ratio of wet/dry weight in the saline-treated mice after LPS exposure was significantly increased (P < 0.05) compared with that measured in LPS-exposed mice treated with MLCK inhibitor. *P < 0.05 by Student's t-test.

 
Light microscopic evaluation of lung histology following endotoxin exposure. To examine more closely the combination of endotoxin-induced ALI and VALI after 2 h of protective ventilation, we used semiquantitative methods (29) in the light microscopic evaluation of H&E-stained lung sections (Fig. 6). In these experiments, all mice underwent 2 h of protective mode ventilation after exposure to LPS or saline using the same treatment regimen as used in the assessment of compliance and gas exchange. The lungs of both MLCK KO (Fig. 6A; 4.5 ± 0.3) and WT (Fig. 6B; 5.0 ± 0.5) saline-treated control mice were normal. WT mice treated with endotoxin for 24 h before ventilation showed diffuse, heterogeneous pulmonary injury (Fig. 6, C and D; 15.4 ± 0.5) with widespread vascular congestion with hemorrhage, perivascular edema, and atelectasis. At higher power (Fig. 6D), inflammatory infiltrates were also apparent. In contrast, the sections prepared from lungs of both MLCK KO mice exposed to endotoxin (Fig. 6E; 5.0 ± 0.5) or WT mice treated with MLCK inhibitor after endotoxin exposure (Fig. 6F; 6.5 ± 1.8) were largely normal in appearance and appeared comparable to the saline-treated control animals in each strain (Fig. 6, A and B).


Figure 6
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  		Fig. 6. MLCK gene KO or treatment with MLCK inhibitor attenuates endotoxin-induced histological lung injury after 2 h of protective ventilation. Saline-treated control KO (A) and WT (B) mice show no atelectasis, minimal inflammatory infiltrate, and minimal perivascular edema. LPS-treated WT mice (C) developed hemorrhage, perivascular edema, and disruption of architecture. High power (D) shows increased inflammatory infiltrate and vascular congestion. Both LPS-treated KO mice (E) and LPS-treated WT mice treated with inhibitor (F) show visible improved aeration, minimal hemorrhage, inflammatory infiltrate, and cellular infiltrate with mild perivascular edema. Bar, 100 µm (A, B, C, E, F). Bar, 50 µm (D).

 
Dose-dependent effects of MLCK inhibitor on compliance changes following endotoxin exposure. To determine the dose dependence of the lung protective properties demonstrated in the previous experiments by treatment with the MLCK inhibitor, we treated WT mice with a range of doses of the inhibitor using the same experimental design as that employed for assessment of changes in compliance and gas exchange. Mice were treated with a single dose of inhibitor 4 h after initial exposure to LPS, and lung compliance was measured after 24 h of exposure to endotoxin and 2 h of the protective mode of ventilation. Inhibitor doses of 2.5, 1.5, 0.75, and 0.25 mg/kg were tested for their ability to attenuate the loss in lung compliance associated with endotoxin exposure (Fig. 7). All inhibitor-treated groups except the lowest dose of inhibitor (0.25 mg/kg; 0.873 ± 0.029) showed a significant improvement in compliance compared with mice treated with LPS alone (0.893 ± 0.039). There was no significant difference in compliance between the two highest doses, 2.5 mg/kg (1.139 ± 0.036) and 1.5 mg/kg (1.109 ± 0.029). In addition, the difference in compliance curves between the 2.5-mg/kg group (1.139 ± 0.036) and the 0.75-mg/kg group (0.921 ± 0.012) was significant (P < 0.05).


Figure 7
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  		Fig. 7. Dose effects of MLCK inhibitor on changes in lung compliance associated with endotoxin exposure. WT mice (n = 6 for each group) were treated with 10 mg/kg LPS, followed by inhibitor at 1 of 4 doses (0.25, 0.75, 1.5, or 2.5 mg/kg) 4 h after LPS. The slope of the compliance curve was not significantly different between WT mice exposed to LPS (bullet) (0.893 ± 0.039) and WT+LPS mice treated with 0.25 mg/kg of inhibitor ({square}) (0.873 ± 0.029). Mice treated with LPS and either 0.75 mg/kg (0.921 ± 0.012) ({lozenge}), 1.5 mg/kg (1.109 ± 0.029) ({triangleup}), or 2.5 mg/kg (1.139 ± 0.036) ({blacktriangledown}) of inhibitor showed a significant (P < 0.5) increase in the slope of the compliance curve compared with WT+LPS or WT+LPS+0.25 mg/kg of inhibitor. The slope of the compliance curves between the 0.75-mg/kg group and the 2 higher doses was significantly different (P < 0.05). There was no significant difference in the slope of the 1.5-mg/kg and the 2.5-mg/kg doses. Values are means ± SE.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The insults used in this study, endotoxin, mechanical ventilation, and 1.00 FIO2, represent frequent causes of injury and complications of therapy in critical care medicine. The convergent gene KO and chemical biology results of these studies are the first in vivo demonstration of a link between MLCK and the fundamental lung function of gas exchange. The proximity of endothelial to epithelial cells forms the barrier between the gas exchange units of the lung, the alveoli, and the capillary network. MLCK210 is found in both cell types, and disruption of this barrier in the lung results in edema, decreased compliance, disturbed gas exchange, and acidosis. The cardinal role played by MLCK in maintaining both pulmonary barrier integrity and function is supported by the protection afforded against compromised lung function by both MLCK210 gene KO and treatment with an MLCK inhibitor.

The integrity of the pulmonary blood-gas barrier is essential for effective gas exchange (35). Despite exposure to multiple insults including 100% inspired oxygen, endotoxin, and mechanical ventilation, mice in this study showed improved survival, preserved oxygenation, and protection against acidosis. The functional role of MCLK in this protection is supported by the accompanying reduction in edema, preservation of lung function, and the greater survival both in the KO and inhibitor-treated mice. Other signaling pathways (1, 5, 25) implicated in MLCK-mediated regulation of barrier function may also contribute to such protection, and their role remains to be elucidated.

Pulmonary edema can result from impairment of EC barrier integrity or increased hydrostatic pressure associated with cardiac dysfunction (23). The pulmonary system is more susceptible to injury than other tissues due to the vast endothelial vascular bed in the lung (19). Stimulation of endothelial surface receptors (25, 30) results in activation of multiple pathways leading to phosphorylation of MLC. The phosphorylation state of MLC is a key determinant of EC barrier function and the increase in paracellular transport in disease (10, 13, 17). Phosphorylated MLC leads to the creation of actin stress fibers (11), cell contraction, gap formation, and the development of vascular leak. The congruent results obtained from both MLCK210 KO mice and mice treated with the MLCK inhibitor together imply that the protection afforded by selective MLCK210 gene KO or treatment with inhibitor reflect preservation of MLCK function during ALI or ALI combined with VALI.

Gravimetric measurements of lung edema and histological analysis of lung sections with normal architecture along with a normal PaO2/FIO2 ratio and pH also suggest the preservation of lung function afforded by MLCK210 gene KO, or treatment with MLCK inhibitor is due to the maintenance of the endothelial barrier integrity. The elevation of the hematocrit in the endotoxin-exposed WT mice most probably reflects heme concentration due to dehydration and capillary leak and is consistent with perivascular edema observed in the lung H&E sections. However, this result could also be due to a more diffuse capillary leak that would be easier to differentiate in a larger animal.

To obtain end points directly relevant to the clinical management of critically ill patients and to link MLCK to compromise of pulmonary physiology in vivo, we delivered the MLCK inhibitor in a chemotherapeutic, delayed administration protocol. The time frame of inhibitor administration, 4 h after endotoxin exposure, includes the phase of maximal cytokine expression in endotoxin-treated mice (15) and is therefore likely to be associated with the maximal cytokine and inflammation-induced EC leakage (24). The delayed administration of MLCK inhibitor was intended to recapitulate the relative order of patient presentation and initiation of therapy.

Established protective mechanical ventilation regimens are used to minimize, but do not prevent, iatrogenic lung injury (7, 34, 36). In the present study, treatment with the MLCK inhibitor afforded an additional level of protection against loss of lung compliance beyond that provided by a standard protective ventilation regimen. These data suggest that treatment with the MLCK inhibitor improves lung function associated with ALI and iatrogenic VALI and imply a potential role for the use of an MLCK inhibitor as a cotherapy with standard clinical practices in the management of patients requiring mechanical ventilation.

For this compound to be considered as potential drug treatment, there should be a dose-dependent effect. We were able to show a trend toward dose dependence with the compliance curves showing significant differences between three of the five doses of inhibitor administered. A true dose-dependent challenge using these outcome measures would require evaluation in a larger species.

The small molecule MLCK inhibitor used in these studies does not distinguish between the two MLCK isoforms. Nevertheless, the effect of this treatment most likely reflects interaction of the inhibitor with MLCK210. The inhibitor, based on a privileged aminopyridazine scaffold (18), shows selective inhibition of MLCK in vitro compared with other calcium calmodulin-regulated protein kinases (31) and targets the enzyme catalytic domain that is common to both MLCK210 and MLCK108. MLCK210 is more abundant in EC tissue compared with MLCK108 (4). The relative specificity and selectivity in vivo of the inhibitor for MLCK210 function reflects the target tissue distribution of MLC210, the intrinsic properties of the pulmonary endothelium, and the dose of the inhibitor used.

The investigations summarized here have four key findings. First, we demonstrate an in vivo linkage between EC MLCK210 and lung function as measured by preserved gas exchange. Second, we use a novel small molecule MLCK inhibitor to show an in vivo protective effect of MLCK inhibition on the loss of lung function and mortality associated with endotoxin-induced ALI combined with VALI. Third, we show that MLCK inhibitor treatment can be effective in a clinically relevant time window after initiation of endotoxin responses. Fourth, we show that attenuation of lung injury by MLCK inhibitor treatment is complementary to the use of a standard clinical protective mechanical ventilation strategy. The use of clinically relevant end points bridges the gap between bench research and clinical medicine and facilitates further studies of the mechanisms of barrier dysfunction and the potential of MLCK as a therapeutic target.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported in part by National Institute of Neurological Disorders and Stroke Grants R01-NS-047586 (D. M. Watterson) and KO8-NS-044998 (M. S. Wainwright), The Center for Drug Discovery and Chemical Biology (D. M. Watterson), and The American Academy of Pediatrics, Section of Critical Care New Investigator Award (J. L. Rossi). J. L. Rossi and A. V. Velentza were postdoctoral trainees in the Northwestern Drug Discovery Training Program supported by National Institute on Aging Training Grant T32-AG-00260.


   ACKNOWLEDGMENTS
 
We thank C. Stephens and J. Schavocky for technical assistance and Dr. Susan Crawford for advice and expertise with analysis of histological specimens. Present address of A. V. Velentza: Genomics Institute of the Novartis Research Foundation, San Diego, CA.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. L. Rossi, Northwestern Univ., 303 E. Chicago Ave., Ward 8-196, Chicago, IL 60611 (e-mail: j-rossi{at}northwestern.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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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