© 2003 American Thoracic Society
The Inducible Nitric Oxide Synthase Inhibitor BBS-2 Prevents Acute Lung Injury in Sheep after Burn and Smoke Inhalation InjuryDepartments of Anesthesiology and Pathology, University of Texas Medical Branch, and Shriners Burns Hospital, Galveston, Texas; and Departments of Medicinal Chemistry and Immunology, Berlex Biosciences, Richmond, California Correspondence and requests for reprints should be addressed to Daniel L. Traber, Ph.D., Department of Anesthesiology, UTMB, 610 Texas Ave, Galveston, TX 77555. E-mail: dltraber{at}utmb.edu
In this study we examined the role of inducible nitric oxide synthase (iNOS) in acute respiratory distress syndrome (ARDS) in sheep with severe combined burn and smoke inhalation injury. BBS-2, a potent and highly selective iNOS dimerization inhibitor, was used to exclude effects on the endothelial and neuronal NOS isoforms. Seven days after surgical recovery, sheep were given a burn (40% of total body surface, 3rd degree) and insufflated with cotton smoke (48 breaths, < 40°C) under anesthesia. BBS-2 was provided by constant infusion at 100 µg/kg/hour, beginning 1 hour after injury. During 48 hours, control sheep developed multiple signs of ARDS. These included decreased pulmonary gas exchange, increased pulmonary edema, abnormal lung compliance, and extensive airway obstruction. These pathologies were associated with a large increase in tracheal blood flow and elevated plasma NO2-/NO3- (NOx) levels. These variables were all stable in sham animals. Treatment of injured sheep with BBS-2 attenuated the increases in tracheal blood flow and plasma NOx levels, and significantly attenuated all the pulmonary pathologies that were noted. The results provide definitive evidence that iNOS is a key mediator of pulmonary pathology in sheep with ARDS resulting from combined burn and smoke inhalation injury.
Key Words: nitric oxide • acute respiratory distress syndrome • thermal injury Despite effective fluid resuscitation management and early surgical excision of burned tissue, the mortality rate of combined burn and smoke inhalation injuries is still high. Patients with extensive cutaneous burns in which the burned area exceeds 30% of the total body surface area exhibit microvascular pressure and hyperpermeability increases, not only at the injured site but also in regions distant from the injury, leading eventually to burn shock (1, 2). The pulmonary vascular hyperpermeability leads to lung edema formation (3, 4). All of the burn related pathology appears to be more severe if the thermal damage is combined with inhalation injury (5, 6). Smoke inhalation is a major determinant in the mortality of fire victims. Inhalation injury alone increases mortality by a maximum of 20% and pneumonia by a maximum of 40% (7). Inflammatory exudates and aggregates of mucus, sloughed epithelial mucosa and cellular debris, together with impaired mucociliary clearance, lead to airway obstruction. Airway obstruction and pulmonary edema are accompanied by impairment of respiratory gas exchange and bacterial colonization of the lungs with subsequent development of tracheobronchitis, atelectasis, and pneumonia leading to acute respiratory distress syndrome (ARDS), one of the major complications in fire victims with combined burn and smoke inhalation injuries. It is well known that nitric oxide (NO) plays an important role in the pathogenesis of conditions that are frequently complicated by ARDS, such as sepsis and multiple trauma, including burn and smoke inhalation injuries (8, 9). NO is formed from arginine by the enzyme nitric oxide synthase (NOS). There are three isoforms of NOS. One isoform, an inducible nitric oxide synthase (iNOS), is induced by cytokines and bacterial products (10, 11). These cytokines and bacteria are present in multiple trauma, including the combination of burn and smoke inhalation. Recently, our laboratory has shown the involvement of iNOS in the pathogenesis of cardiopulmonary pathology and ARDS in sheep with combined burn and smoke injury (12). This evidence was initially obtained with L-NG-nitroarginine methyl ester, a nonselective NOS inhibitor, and subsequently with mercaptoethylguanidine (MEG), a weak and only partially selective iNOS inhibitor (12). Because of the lack of truly selective NOS inhibitors, it is still unclear which isoform of NOS is principally involved in the pathogenesis of burn/smoke-related acute lung injury. There are no reports of the effect of truly selective iNOS inhibitors on the lesions of ARDS, particularly in the large animal models, that allow us to measure the cardiopulmonary changes for long-term experiments. In the present report we aimed to clarify the role of iNOS in acute lung injury in sheep after combined burn and smoke inhalation injuries by using BBS-2, a potent and highly selective pyrimidylimidazole-based iNOS dimerization inhibitor (13, 14).
This study was approved by the Animal Care and Use Committee of the University Texas Medical Branch. All the animals were handled according to the guidelines established by the American Physiology Society and the National Institutes of Health.
Animal Model Twenty-two adult female sheep (30–40 kg) were surgically prepared as previously described (16). After a 5-day recovery period, the sheep were anesthetized with halothane and given a burn (40% of total body surface area, 3rd degree) and inhalation injury. The third degree of burn is not associated with pain because the nerve endings in tissue will be destroyed during the injury (16). Inhalation injury was induced with a modified bee smoker (12). After the injury, all animals were maintained on mechanical ventilation (Servo Ventilator 900C; Siemens-Elema, Solna, Sweden) throughout the 48-hour experimental period. Ventilation was performed with a positive end-expiratory pressure of 5 cm H2O and a tidal volume of 15 ml/kg. During the first 3 hours after injury, the inspiratory O2 concentration was maintained at 100%, and respiratory rate was maintained at 30 breaths/minute. Then both were adjusted according to blood gas analysis to maintain arterial O2 saturation above 90% and PCO2 between 25–30 mm Hg. All animals are resuscitated with Ringer's solution according to Parkland formula (4 ml/kg/% burned body surface for the first 24 hours, and 2 ml/kg/% burned body surface for the second 24 hours [12]). However, for the first 8 hours after the injury, the sheep received one half of fluid was given for first 24 hrs. The sheep were divided randomly into four groups: sham (n = 6), sham/BBS-2 (n = 4), control (n = 6) (injured, but not treated), and BBS-2 (n = 6) (injured, but treated with BBS-2). The animals were randomized before the injury. After randomization, baseline data were obtained, and then animals were given burn and smoke inhalation injury in the same manner. For treatment we used a new potent and selective iNOS inhibitor, BBS-2 (ZK-809984; Berlex, Richmond, CA). The IC50 of BBS-2 for human iNOS is 0.49 nM in the human cell line A-172 (14). In dimerization assays, BBS-2 exhibits selectivity for iNOS versus neuronal NOS and endothelial NOS dimerization of 620- and 1,500-fold, respectively (14). Continuous infusion of BBS-2 was begun 1 hour after the insult (100 µg/kg/hour for 48 hours). However, one of the six animals treated with BBS-2 received double dose during the second 24 hours, but it did not cause noticeable cardiopulmonary changes. Control animals received vehicle.
Measured Variables Forty-eight hours after burn/smoke, the sheep were killed under deep isoflurane anesthesia. The right lung was removed, and a 1-cm thick section was taken through the middle of lower lobe. This section was inflated with 10% formalin for histologic examination. Fixed samples were embedded in paraffin, sectioned into 6-µm pieces, and stained with hematoxylin-eosin (H-E). One hundred bronchi, bronchioles, and respiratory bronchioles were evaluated and the percentage of area obstructed by the cast was estimated (0–100%). The remaining lower half of the right lower lobe was used for determination of wet-to-dry weight ratio. Lung wet-to-dry weight ratio was calculated as an index of lung water content (18).
Statistical Analysis
All animals survived after the combined injury with 40% (total body surface area) burn and smoke inhalation during the 48-hour experimental period. Fluid resuscitation strictly followed the Parkland formula (4 ml/kg/% burned body surface for the first 24 hours, and 2 ml/kg/% burned body surface for the second 24 hours [12]). Body weight of BBS-2 and control group was similar (36.5 ± 1.5 and 37.1 ± 1.5 kg, respectively). The arterial carboxyhemoglobin levels immediately after smoke exposure were 58.8 ± 4.9% in BBS-2–treated group and 61.2 ± 5.0% in the control group. There was no statistical difference (p = 0.79) between these values, indicating that both control and treated animals received similar injuries. There were also no statistical differences between baseline values of both treated and untreated group of animals (Heart rate, 89 ± 3.3 and 88 ± 3;  , 104.5 ± 9.5 and 98 ± 2.5; Pulmonary arterial pressure (Ppa), 20 ± 0.3 and 19.1 ± 0.4; lung lymph flow, 5.2 ± 0.7 and 6.4 ± 0.2 in treated and untreated groups, respectively). Injection of BBS-2 alone (without injury) into sham animals did not affect the cardiopulmonary parameters (Table 1
, Figures 1–3)
.
Effect of BBS-2 on Plasma Concentration of Nitrite and Nitrate Plasma NOx levels were stable in sham animals. Animals with combined burn and smoke inhalation injury showed 2- and 2.5-fold increases in plasma NOx levels at 24 and 48 hours, respectively. The increase in plasma NOx levels seen in injured animals was completely inhibited by infusion of 100 µg/kg/hour BBS-2, suggesting involvement of iNOS-derived NO (Figure 1).
Effect of BBS-2 on Pulmonary Gas Exchange and Shunt Fraction
Effect of BBS-2 on Increase in Lung Lymph Flow
Effect of BBS-2 on Lung Water Content
Effect of BBS-2 on Airway Obstruction A major sequelae of the burn and smoke inhalation injury is airway cast formation. In the present study, we determined airway obstruction scores in bronchi, bronchioles, and respiratory bronchioles of all three groups of animals. The sham group of animals showed little evidence of airway obstruction: 6.9, 2.5, and 0.17% of bronchi, bronchioles, and respiratory bronchioles, respectively. The control group of injured animals showed high airway obstruction scores: 24.5, 13.5, and 2.8% of bronchi, bronchioles and respiratory bronchioles, respectively (Figure 5) . Inhibition of iNOS by BBS-2 resulted in significant inhibition of airway obstruction: 9.3, 2.8, and 0.6% of bronchi, bronchioles, and respiratory bronchioles, respectively, were obstructed.
Effect of BBS-2 on Airway Blood Flow Airway (tracheal) blood flow was immediately increased after injury in control animals, peaking at 12 hours and remaining elevated throughout the experimental period (Figure 6) . BBS-2 markedly reduced this increase in tracheal blood flow.
Effect of BBS-2 on Airway Pressures The airway pressures (peak and pause airway pressure) were markedly increased in control animals beginning from 24 hours after insult. The baseline values of peak and pause pressures were 21.6 ± 1.0 and 19.2 ± 0.9, respectively. These numbers were almost doubled at 48 hours after insult: 42.8 ± 4.8 (p < 0.05) and 36.8 ± 3.8 (p < 0.05) for the peak and pause pressure, respectively. Treatment with BBS-2 reduced these increases in both peak (Figure 7A) and pause pressure (Figure 7B) almost to the baseline levels.
Effect of BBS-2 on Cardiovascular Variables Control animals showed significant increase in heart rate (36, 48 hours), Ppa (24, 36, 48 hours), and capillary pressure (24, 36 hours). Treatment with BBS-2 did not adversely affect these variables. There was no significant difference between groups for (Table 1).
ARDS is a serious complication in fire victims with burn and smoke inhalation injuries. The progressive respiratory failure associated with lung edema is an important determinant of mortality. In these patients, the lung is the first target organ, because the blood from burned tissue passes the pulmonary circulation before passing other organs (19). The lung vascular leakage leads to a large amount of fluid flux from circulating blood into pulmonary interstitial space, and the consequent edema formation is more severe when thermal injury is associated with smoke inhalation injury (5, 6). In the present study, the control animals showed typical cardiopulmonary response to burn/smoke injury (15, 20). The pathologic changes in control animals were characterized by a marked increase in pulmonary vascular permeability with formation of lung tissue edema. There was also a drastic increase in bronchial blood flow associated with airway edema and cast formation. The alveolar and airway edema resulted in poor pulmonary gas exchange. Although the mechanism of these alterations remain unclear, the inhibition of NO prevents these changes, suggesting a possible participation of NO in pathogenesis of burn/smoke-related lung injury. With combined thermal and inhalation injuries, cytokines such as interleukin-1 are upregulated in lung tissue (21). In addition, translocation of endotoxin and bacteria from the intestine into the systemic circulation has been reported to occur after burn/smoke injury (22, 23). Both interleukin-1 and endotoxin upregulate iNOS, which catalyzes production of large amounts of NO, and under conditions of substrate or cofactor limitation, may also synthesize O2- (24). The formation of NO in the lung results in the loss of hypoxic vasoconstriction (25). NO also can combine with O2- to form the highly oxidative material peroxynitrite (ONOO-) (26, 27). The peroxynitrite may damage the alveolar capillary membrane (28), resulting in additional pulmonary edema. We have previously reported the presence of peroxynitrite in the airway and parenchyma of the lung in sheep after burn and inhalation injury (12).
We have previously shown some beneficial effects of nonspecific NOS inhibitors on ARDS in sheep with combined burn and smoke inhalation injuries. L-NG-nitroarginine methyl ester significantly attenuated the decrease in pulmonary gas exchange and reduced arginine uptake by the lung (unpublished data, K. Murakami and colleagues). On the other hand, L-NG-nitroarginine methyl ester showed some unexpected negative effects such as increase in In this study, we tested the effect of new potent and selective iNOS inhibitor, BBS-2, that inhibits the dimerization of the iNOS enzyme. Dimerization of iNOS is initiated by heme insertion (32, 33), and in the presence of tetrahydrobiopterin (H4B) and L-arginine, the enzyme forms a stable, active dimer. BBS-2 coordinates the heme in the iNOS monomer and prevents both arginine and H4B from binding to these sites, leading to the formation of a "dead-end" inhibitor–monomer complex (13). The IC50 of this compound for human iNOS is 0.49 nM in the human cell line A-172 (14). In dimerization assays, BBS-2 exhibits selectivity for iNOS versus nNOS and eNOS dimerization of 620- and 1,500-fold, respectively (14). The effects of BBS-2 in the model can thus be confidently assigned to selective effects on iNOS inhibition without acute effects on eNOS or nNOS activity.
The current studies focused on the profile of BBS-2 with respect to intrapulmonary pathophysiologic lesions that promote the development of ARDS secondary to combined burn and smoke inhalation injury. Inhibition of iNOS by BBS-2 resulted in significant improvement of pulmonary function without adversely affecting cardiopulmonary hemodynamic parameters, including heart rate, BBS-2 also significantly attenuated the increase in lung lymph flow, which is a direct measure of enhanced pulmonary vascular permeability (37). Wet-to-dry weight ratios and an independent histologic score for pulmonary edema corroborated the effects of BBS-2 on lung lymph flow. As we mentioned, all group of animals including sham received the same amount of fluid, according to the Parkland formula. In addition, BBS-2 did not affect the capillary pressure and Ppa (Table1). Thus the attenuating effect of BBS-2 on pulmonary edema formation is unlikely due to difference in hydrostatic pressure. This burn/smoke-related lung injury might be due to inflammation and cytokines such as interleukin-1, which causes the activation of leukocytes. The toxic product, a superoxide radical released from these activated neutrophils, can form peroxinitrite, a more toxic substance in presence of NO. Inhibition of excessive NO by BBS-2 may result in prevention of peroxynitrite formation, thereby in less lung injury. Overall the evidence with BBS-2 suggests a profound contribution of iNOS to airway injury via several mechanisms that are triggered by iNOS expression in multiple cell types. The results suggest a complex interplay between iNOS-derived NO, O2-, and peroxynitrite with other inflammatory mediators in promoting cellular dysfunction. These integrated effects of iNOS contribute to pathophysiology-enhanced airway blood flow and inflammatory cell infiltration, mucus secretion and fibrin-rich cast formation, ventilation/perfusion mismatch, and microvascular dysfunction leading to pulmonary edema. These processes would all contribute to poor gas exchange and abnormal lung compliance in ARDS. Taken together, our studies show that iNOS plays a significant role as a mediator of intrapulmonary pathology in a clinically relevant sheep model of severe ARDS secondary to combined smoke inhalation and burn injury. The salutary effects of BBS-2 in preventing all aspects of the lung pathology examined and the marked improvements in lung function observed, and absence of negative cardiopulmonary side effects examined, provide a strong rationale that selective iNOS inhibitors should be considered for the prevention and or treatment of ARDS in burn and smoke inhalation injury.
The authors would like to thank David D. Davey (Department of Medicinal Chemistry, Berlex Biosciences, Richmond, CA) for the synthesis and preparation of BBS-2 in support of these studies. Received in original form September 11, 2002; accepted in final form December 20, 2002
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ABSTRACT
METHODS
p < 0.05 versus control, and 
p < 0.05 versus sham/BBS-2.
s/
p < 0.05 versus control (respiratory bronchiole).
