Beneficial pulmonary effects of a metalloporphyrinic peroxynitrite decomposition catalyst in burn and smoke inhalation injury

Abstract

During acute lung injury, nitric oxide (NO) exerts cytotoxic effects by reacting with superoxide radicals, yielding the reactive nitrogen species peroxynitrite (ONOO⁻). ONOO⁻ exerts cytotoxic effects, among others, by nitrating/nitrosating proteins and lipids, by activating the nuclear repair enzyme poly(ADP-ribose) polymerase and inducing VEGF. Here we tested the effect of the ONOO⁻ decomposition catalyst INO-4885 on the development of lung injury in chronically instrumented sheep with combined burn and smoke inhalation injury. The animals were randomized to a sham-injured group (n = 7), an injured control group [48 breaths of cotton smoke, 3rd-degree burn of 40% total body surface area (n = 7)], or an injured group treated with INO-4885 (n = 6). All sheep were mechanically ventilated and fluid-resuscitated according to the Parkland formula. The injury-related increases in the abundance of 3-nitrotyrosine, a marker of protein nitration by ONOO⁻, were prevented by INO-4885, providing evidence for the neutralization of ONOO⁻ action by the compound. Burn and smoke injury induced a significant drop in arterial Po₂-to-inspired O₂ fraction ratio and significant increases in pulmonary shunt fraction, lung lymph flow, lung wet-to-dry weight ratio, and ventilatory pressures; all these changes were significantly attenuated by INO-4885 treatment. In addition, the increases in IL-8, VEGF, and poly(ADP-ribose) in lung tissue were significantly attenuated by the ONOO⁻ decomposition catalyst. In conclusion, the current study suggests that ONOO⁻ plays a crucial role in the pathogenesis of pulmonary microvascular hyperpermeability and pulmonary dysfunction following burn and smoke inhalation injury in sheep. Administration of an ONOO⁻ decomposition catalyst may represent a potential treatment option for this injury.

the mortality rates of patients with severe burn trauma remain high, despite the establishment of standard treatment strategies, including early surgical excision of burned tissue, mechanical respiratory support, and effective fluid-resuscitation management. The presence of concomitant inhalation injury is a major determinant of morbidity and mortality of fire victims (21, 26), contributing to severe lung injury and deterioration of pulmonary function.

Combined burn and smoke inhalation injury in sheep closely reflects the pathophysiological changes in humans with these injuries (10), and previous studies with our model demonstrated that reactive nitrogen species (RNS) are critically involved in this process (5, 24, 28, 36). We have also reported the presence of reactive oxygen species (ROS) in patients with burn and smoke inhalation injury (20). Nitric oxide (NO) combines with superoxide radicals, yielding the RNS peroxynitrite (ONOO⁻) (31). ONOO⁻ exerts deleterious effects by nitrating/nitrosating various other molecules or decaying and producing secondary reactive species such as hydroxyl radicals (29, 30). ONOO⁻ has also been shown to trigger the activation of the nuclear repair enzyme poly(ADP-ribose) (PAR) polymerase (PARP) via DNA strand breakage (11, 34) and upregulation of VEGF (22). All the above-described mechanisms may be critical in the pathogenesis of pulmonary microvascular hyperpermeability and severe lung dysfunction, as typically seen in humans and sheep subjected to thermal injury and smoke inhalation (5, 10, 26).

We hypothesized that ONOO⁻ formation is a major component of the pulmonary damage that occurs with combined burn and smoke inhalation injury, and we determined the effects of a potent ONOO⁻ decomposition catalyst on the pathophysiology of acute lung injury seen with combined burn and smoke inhalation in chronically instrumented sheep.

MATERIALS AND METHODS

This study was approved and monitored by the Institutional Animal Care and Use Committee of The University of Texas Medical Branch. The studies were accomplished in the Investigative Intensive Care Unit, a facility certified by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

Surgical preparation and injury.

Twenty healthy adult female sheep with a mean body weight of 34 ± 2 kg were included in this study. After induction of anesthesia with ketamine (500 mg im, 300 mg iv), endotracheal intubation was performed. Anesthesia was maintained using an isoflurane (1.4–1.8 vol%)-O₂ (50%) mixture. The right femoral artery was cannulated with a polyvinylchloride catheter (16-gauge, 24 in., Intracath, Becton Dickinson Vascular Access, Sandy, UT) for continuous measurement of systemic arterial pressure and intermittent sampling of arterial blood. A thermodilution catheter (model 93A-131-7F, Edwards Critical Care Division, Irvine, CA) was inserted into the right external jugular vein through an introducer sheath (Edwards Lifescience, Irvine, CA) and advanced into the common pulmonary artery. Through the left fifth intercostal space, a Silastic catheter (0.062 in. ID, 0.125 in. OD; Dow Corning, Midland, MI) was positioned in the left atrium for continuous measurement of left atrial pressure. Through the right fifth intercostal space, a Silastic catheter (0.025 in. ID, 0.047 in. OD) was placed into an efferent lymphatic vessel from the caudal mediastinal lymph node for the measurement of lung lymph flow (Q̇_L). The systemic lymph contribution was removed by ligation of the tail of the caudal mediastinal lymph node and cauterization of the the systemic diaphragmatic lymph vessels (5, 17, 28). After a recovery period of 5–7 days, a baseline measurement was performed in spontaneously breathing sheep. Thereafter, the animals were anesthetized using ketamine (5 mg/kg iv). Tracheotomy was performed, and anesthesia was maintained with a halothane (1.1–2.0 vol%)-O₂ (50%) mixture. Under deep anesthesia, the animals were subjected to combined burn and smoke inhalation injury according to an established protocol that was previously described in detail (5, 17, 28). The animal's wool was removed, and each flank was subjected to a 20% total body surface area (TBSA) third-degree (full-thickness) flame burn. Inhalation injury was induced using a modified bee smoker filled with 40 g of burning cotton toweling and connected to the tracheostomy tube via a modified endotracheal tube containing a thermistor (13). During the insufflation procedure, the temperature of the smoke was monitored carefully and not allowed to exceed 40°C. The sheep was insufflated with a total of 48 breaths (4 sets of 12 breaths each) of cotton smoke. Arterial carboxyhemoglobin concentrations were determined immediately after each set of smoke inhalation exposures (CO-Oximeter 282, Instrumentation Laboratory, Lexington, MA). A Foley urinary retention catheter was inserted. Anesthesia was then discontinued, and the sheep were allowed to awaken.

Experimental protocol.

After injury, the sheep were randomly allocated to the following three study groups: 1) sham-injured, nontreated animals [sham (n = 7)], 2) injured, nontreated animals [control (n = 7)], and 3) injured animals treated with the metalloporphyrinic ONOO⁻ decomposition catalyst INO-4885 {[5,10,15,20-tetra[N-(benzyl-4′-carboxylate)-2-pyridinium]-21H,23H-porphine iron(III) chloride]; kindly provided by Inotek Pharmaceuticals, Beverly, MA (n = 6)} (12). INO-4885 is a pyridyl-substituted porphyrin that catalyzes the degradation of hydrogen peroxide, nitroxyl anions, and OONO⁻ to benign species with a reaction rate of ∼10⁷ M/s. At 1 h after the injury, a bolus of 0.5 mg/kg INO-4885 (dissolved in saline) was intravenously administered and then continuously infused intravenously at 0.01 mg·kg⁻¹·h⁻¹ for the remainder of the 24-h observation period. The dose was selected on the basis of 1) previous range-finding studies in rodents in myocardial ischemia-reperfusion (12), 2) considerations with respect to differences in pK and metabolism between rodents and large animals, and 3) preliminary dose-finding studies in sheep. Sham and control animals received an equivalent volume of the saline vehicle.

All sheep were mechanically ventilated (Servo Ventilator 900C, Siemens, Elema, Sweden) with a tidal volume of 12–15 ml/kg and a positive end-expiratory pressure of 5 cmH₂O. A higher tidal volume is required for sheep than for humans, because sheep have a higher dead space-to-tidal volume ratio (0.57 vs. 0.30) (33). The inspired O₂ fraction (Fi_O2) was set at 1.0 for the first 3 h after injury to induce rapid carboxyhemoglobin clearance and then adjusted to maintain sufficient oxygenation [>90% arterial O₂ saturation, 80–100 mmHg arterial Po₂ (Pa_O2)] whenever possible. The respiratory rate was initially set at 20 breaths/min and then adjusted according to blood gas analyses to maintain the arterial Pco₂ within 5 mmHg of the baseline value. All animals were fluid-resuscitated with lactated Ringer solution according to the Parkland formula (4 ml·kg⁻¹·%TBSA⁻¹·24 h⁻¹). All animals had free access to dry food, but not water, to control the fluid balance. At the end of the 24-h study period, the animals were deeply anesthetized with ketamine (15 mg/kg) and euthanized by intravenous injection of 60 ml of saturated potassium chloride.

Hemodynamic measurements.

Mean arterial pressure (MAP), central venous pressure (CVP), left atrial pressure (LAP), mean pulmonary arterial pressure (MPAP), pulmonary capillary wedge pressure (PCWP), and heart rate (HR) were determined from the femoral and pulmonary artery catheters using pressure transducers (Baxter-Edwards Critical Care, Irvine, CA) and recorded on a hemodynamic monitor (model V24C, Philips Medicine System Bollinger, Bollinger, Germany). Cardiac output was measured in triplicate with the thermodilution technique (model 9530, Baxter-Edwards Critical Care). Cardiac index (CI) was used as a measure of cardiac output related to body surface area. Stroke volume index (SVI) was calculated as CI/(HR × 1,000). Left ventricular stroke work index (LVSWI) was calculated as (MAP − LAP) × SVI × 0.0136. Systemic vascular resistance index (SVRI) was determined as (MAP − CVP)/(CI × 80) and pulmonary vascular resistance index (PVRI) as (MPAP − PCWP)/(CI × 80).

Blood analysis.

Blood gases were measured using a blood gas analyzer (Synthesis 15, Instrumentation Laboratories, Lexington, MA). The Pa_O2-to-Fi_O2 ratio, pulmonary shunt fraction (Qs/Qt), and vascular resistances were calculated using standard equations. Blood cell count was performed with a Hemavet 850 (Drew Scientific, Oxford, CT) adjusted for sheep blood. In addition, blood was centrifuged, and plasma and serum samples were frozen at −80°C for the determination of serum aspartate aminotransferase, alanine aminotransferases, bilirubin, and creatinine (Vitros 5.1 FS, Ortho Clinical Diagnostics, Rochester, NY). Creatinine clearance (ml/min) was estimated as follows: urine creatinine concentration (mg/dl) × urine volume (mol)/serum creatinine concentration (mg/dl) × collection time (min). Plasma colloid oncotic pressure (π_P) was determined with a colloid osmometer (model 4420, Wescor, Logan, UT). Plasma protein concentration (C_P) was measured with a refractometer (National Instrument, Baltimore, MD). NO levels were evaluated by measurement of the intermediate and end products (NOx) using a nitrate/nitrite colorimetric assay kit (Cayman Chemicals, Ann Arbor, MI).

Lymph measurements.

Lung Q̇_L was measured with graduated test tubes and a stopwatch. Lymph colloid oncotic pressure (π_L), lymph protein concentration (C_L), and lymph NOx were measured with the same methods used for plasma measurements. For estimation of pulmonary microvascular permeability to protein, the C_L-to-C_P ratio (C_L/C_P) and π_L-to-π_P ratio (π_L/π_P) were calculated (18). In addition, lymph protein clearance (LPC) was calculated as follows: LPC = Q̇_L × C_L/C_P (23).

Tissue analysis.

After completion of the 24-h experiment, the right lung was removed, and from the middle of the lower lobe, bloodless lung wet-to-dry weight ratio was calculated as an index of lung water content. The following analyses were performed in tissue from five animals per group.

For immunoblotting analyses, lung tissue was immediately frozen in liquid nitrogen and stored at −80°C. Protein expression of 3-nitrotyrosine (3-NT; mouse monoclonal 3-NT antibody; catalog no. 39B6, Abcam, Cambridge, MA), PAR (mouse monoclonal anti-PAR; clone 10H, Millipore, Billerica, MA), IL-8 (mouse monoclonal IL-8; catalog no. B-2, Santa Cruz Biotechnology, Santa Cruz, CA), and VEGF (mouse monoclonal VEGF antibody; catalog no. VG-1, Abcam) was measured in lung tissue homogenates using a Western blot protocol, as described previously (16). Blots were quantified by National Institutes of Health IMAGEJ scanning densitometry and normalized to total actin expression. The activity of myeloperoxidase (MPO), an indicator of neutrophil accumulation, was determined directly in lung homogenates. MPO concentrations were evaluated on homogenized whole lungs with a commercially available assay (CytoStore, Calgary, AB, Canada). MPO activity was normalized to lung wet-to-dry weight ratio and reported as units per gram of dry tissue, as previously reported (16).

For the immunohistochemical detection of PAR, monoclonal anti-PAR antibody (Calbiochem, San Diego, CA; 1:1,000 dilution, overnight, 4°C) was used after antigen retrieval. Secondary labeling was achieved by using biotinylated horse anti-mouse antibody (Vector Laboratories, Burlingame, CA; 30 min, room temperature). Horseradish peroxidase-conjugated avidin (30 min, room temperature) and brown-colored diaminobenzidine (6 min, room temperature) were used to visualize the labeling (Vector Laboratories). The sections were counterstained with hematoxylin (blue color). The intensity of PAR staining of individual sections was determined by a blinded experimenter according to a semiquantitative PAR positivity score of 1–10, where 1 = no staining, 2 = light cytoplasmic staining, 3 = few positive nuclei, 4 = light nuclear staining in ∼10% of cells, 5 = light nuclear staining in ∼25% of cells, 6 = light nuclear staining in ∼50% of cells, 7 = strong nuclear staining in ∼50% of cells, 8 = ∼75% positive nuclei, 9 = ∼90% positive nuclei, and 10 = few negative cells (8).

Statistical analysis.

Values are means ± SE. A two-way ANOVA for repeated measurements with appropriate Student-Newman-Keuls post hoc comparisons was used to compare differences within and between groups. One-way ANOVA was used to compare groups when measurements were made at only one time period, and the Newman-Keuls procedure was used for post hoc pairwise comparisons. P < 0.05 was regarded as statistically significant.

RESULTS

There were no differences among study groups in any of the investigated variables at baseline. All animals survived the experimental period.

Organ function parameters.

The injury induced a severe impairment in respiratory gas exchange in the control group, as indicated by a decline in the Pa_O2-to-Fi_O2 ratio and a concomitant increase in Qs/Qt. These alterations were significantly attenuated by treatment with INO-4885 (Fig. 1). The injury-related increases in ventilatory pressures in control animals were also significantly attenuated in the INO-4885 group (Fig. 2). The injury was further associated with a transient increase in serum creatinine concentration and decrease in creatinine clearance, with no differences between INO-4885-treated and control animals. Serum aspartate aminotransferase, alanine aminotransferase, and bilirubin concentrations equally increased over time in both injured groups (data not shown).

Fig. 1.

Impact of peroxynitrite (ONOO⁻) decomposition catalyst INO-4885 on arterial Po₂ (Pa_O2)-to-fraction of inspired O₂ (Fi_O2) ratio (A) and pulmonary shunt fraction (Qs/Qt, B) in sheep with combined burn and smoke inhalation injury. Values are means ± SE. †P < 0.05 vs. sham. *P < 0.05, INO-4885 vs. control.

Fig. 2.

Impact of ONOO⁻ decomposition catalyst INO-4885 on ventilatory pressures [peak airway pressure (A) and pause airway pressure (B)] in sheep with combined burn and smoke inhalation injury. Values are means ± SE. †P < 0.05 vs. sham. *P < 0.05, INO-4885 vs. control.

Pulmonary and systemic microvascular permeability.

The injury led to an increase in pulmonary transvascular fluid flux in the control group, as evidenced by a 10-fold increase in Q̇_L. The increase in Q̇_L was significantly attenuated in the INO-4885 group (Fig. 3A). Lung wet-to-dry weight ratio, a marker of lung water content, was significantly increased vs. sham-injured animals in the control group, but not in the INO-4885 group (Fig. 3B). The urine output was higher in the INO-4885 group than in the control group, but there was no significant difference in net fluid balance between injured groups. Despite identical fluid resuscitation in all study groups, hematocrit and hemoglobin concentrations were significantly elevated in the control group, while these variables remained at baseline levels in INO-4885-treated animals. The injury was associated with marked decreases in C_P and π_P in the control group, which were partially attenuated in the INO-4885 group. There were no group differences in C_L/C_P. However, π_L/π_P was significantly higher in control than in INO-4885-treated animals at 18 h after injury, and the injury-related increase in lymph protein clearance was significantly attenuated by the treatment from 3 to 24 h (Table 1).

Fig. 3.

Impact of ONOO⁻ decomposition catalyst INO-4885 on lung lymph flow (Q̇_L, A) and bloodless lung wet-to-dry weight ratio (B) in sheep with combined burn and smoke inhalation injury. Values are means ± SE. †P < 0.05 vs. sham. *P < 0.05, INO-4885 vs. control.

Table 1.

Changes in protein concentrations, oncotic pressures, urine output, fluid balance, hematocrit, and hemoglobin

	Time After Injury, h
	0	3	6	12	18	24
CP, g/dl
Sham	4.9 ± 0.2	4.6 ± 0.2	4.7 ± 0.2	4.6 ± 0.1	4.8 ± 0.2	4.9 ± 0.2
Control	4.9 ± 0.3	3.7 ± 0.2*	3.6 ± 0.2*	3.2 ± 0.2*	2.7 ± 0.2*	2.7 ± 0.2*
INO-4885	5.1 ± 0.1	3.8 ± 0.2*	3.8 ± 0.1*	3.8 ± 0.1*†	4.0 ± 0.2*†	3.8 ± 0.1*†
CL, g/dl
Sham	3.5 ± 0.3	3.2 ± 0.2	3.0 ± 0.3	3.4 ± 0.3	3.4 ± 0.2	3.1 ± 0.3
Control	3.3 ± 0.1	2.6 ± 0.2	2.1 ± 0.1*	2.0 ± 0.0*	1.8 ± 0.1*	1.6 ± 0.1*
INO-4885	3.9 ± 0.2	2.9 ± 0.2	2.4 ± 0.3	2.5 ± 0.2*	2.4 ± 0.3*	2.3 ± 0.3*†
πP, mmHg
Sham	23.4 ± 1.0	21.8 ± 1.0	22.1 ± 0.9	23.2 ± 1.1	22.8 ± 1.2	22.7 ± 0.8
Control	21.5 ± 0.6	15.3 ± 0.8*	14.4 ± 0.6*	13.4 ± 0.4*	12.2 ± 0.6*	10.7 ± 0.7*
INO-4885	23.1 ± 0.4	16.6 ± 0.6*	16.4 ± 0.9*	16.6 ± 1.0*†	16.1 ± 1.1*†	15.2 ± 1.2*†
πL, mmHg
Sham	13.9 ± 0.12	11.8 ± 1.0	11.8 ± 0.8	13.1 ± 1.1	13.4 ± 0.7	13.0 ± 0.7
Control	13.6 ± 0.5	10.6 ± 0.8	9.3 ± 0.6	9.1 ± 0.3*	8.8 ± 0.5*	6.9 ± 0.3*
INO-4885	15.0 ± 0.9	11.4 ± 0.7	9.5 ± 0.8	10.4 ± 0.9*	9.8 ± 0.8*	9.4 ± 1.0*†
CL/CP
Sham	0.72 ± 0.03	0.71 ± 0.04	0.66 ± 0.05	0.77 ± 0.06	0.74 ± 0.03	0.66 ± 0.04
Control	0.68 ± 0.03	0.71 ± 0.06	0.60 ± 0.04	0.64 ± 0.04	0.69 ± 0.06	0.61 ± 0.07
INO-4885	0.76 ± 0.04	0.77 ± 0.06	0.63 ± 0.06	0.65 ± 0.05	0.60 ± 0.05	0.61 ± 0.06
πL/πP
Sham	0.61 ± 0.04	0.57 ± 0.04	0.56 ± 0.03	0.59 ± 0.04	0.62 ± 0.03	0.60 ± 0.03
Control	0.63 ± 0.02	0.69 ± 0.03*	0.64 ± 0.02	0.68 ± 0.02	0.74 ± 0.06*	0.66 ± 0.02
INO-4885	0.69 ± 0.03	0.69 ± 0.02*	0.58 ± 0.03	0.62 ± 0.03	0.61 ± 0.04†	0.62 ± 0.04
LPC, ml/h
Sham	4 ± 0	4 ± 1	3 ± 0	4 ± 1	4 ± 1	3 ± 1
Control	4 ± 0	11 ± 3*	18 ± 3*	25 ± 4*	27 ± 3*	34 ± 4*
INO-4885	4 ± 0	4 ± 1†	5 ± 1†	9 ± 2†	10 ± 3†	14 ± 6*†
Urine output, ml/kg
Sham		16.9 ± 3.3	26.4 ± 4.1	33.4 ± 5.6	26.7 ± 4.5	27.7 ± 3.9
Control		5.9 ± 1.3*	10.8 ± 1.2*	19.5 ± 3.1*	5.6 ± 1.0*	5.1 ± 1.5*
INO-4885		8.3 ± 2.4*	7.6 ± 0.9*	18.9 ± 4.1*	15.3 ± 3.3*†	16.0 ± 5.2*†
Net fluid balance, ml/kg
Sham		5.3 ± 1.9	7.5 ± 4.1	11.8 ± 5.5	5.9 ± 3.5	2.3 ± 3.8
Control		16.1 ± 3.1*	35.1 ± 3.8*	54.5 ± 6.8*	71.7 ± 7.8*	86.1 ± 8.9*
INO-4885		20.1 ± 2.3*	43.2 ± 2.6*	67.8 ± 7.0*	79.8 ± 7.0*	88.1 ± 9.7*
Hct, %
Sham	28 ± 1	27 ± 1	26 ± 1	26 ± 1	27 ± 1	26 ± 1
Control	28 ± 1	32 ± 2*	31 ± 2*	29 ± 1	32 ± 2*	35 ± 2*
INO-4885	28 ± 1	30 ± 1	28 ± 2	28 ± 2	29 ± 2	29 ± 2†
Hb, g/dl
Sham	10.0 ± 0.2	9.3 ± 0.2	9.1 ± 0.3	9.2 ± 0.3	9.6 ± 0.3	9.5 ± 0.4
Control	9.7 ± 0.3	11.9 ± 0.9*	10.6 ± 0.6	10.1 ± 0.5	10.9 ± 0.6	11.3 ± 0.7*
INO-4885	9.5 ± 0.4	10.6 ± 0.6	9.5 ± 0.5	9.4 ± 0.6	9.6 ± 0.6	9.8 ± 0.5†

Values are means ± SE. C_P, plasma protein concentration; C_L, lymph protein concentration; π_P, plasma colloid oncotic pressure; π_L, lymph colloid oncotic pressure; Hct, hematocrit; LPC, lymph protein clearance.

^*P < 0.05 vs. sham.

^†P < 0.05, INO-4885 vs. control.

Cardiopulmonary hemodynamics.

Mean arterial pressure, systemic vascular resistance index, left atrial pressure, and cardiac index were not different between groups. In the INO-4885 group, stroke volume index and left ventricular stroke work index were significantly higher, while heart rate was significantly lower, than in the control group. The injury led to increases in mean pulmonary arterial pressure and pulmonary vascular resistance index, with no differences between INO-4885-treated and control animals (Table 2).

Table 2.

Changes in cardiopulmonary variables

	Time After Injury, h
	0	3	6	12	18	24
MAP, mmHg
Sham	104 ± 3	113 ± 5	112 ± 3	111 ± 3	103 ± 2	102 ± 2
Control	98 ± 3	109 ± 3	110 ± 3	108 ± 3	98 ± 3	98 ± 5
INO-4885	102 ± 3	110 ± 3	115 ± 4	115 ± 3	113 ± 5	111 ± 6
SVRI, dyn·s·cm−5·m−2
Sham	1,212 ± 49	1,153 ± 60	1,218 ± 68	1,294 ± 64	1,273 ± 83	1,219 ± 52
Control	1,184 ± 59	1,434 ± 122	1,383 ± 105	1,391 ± 87	1,306 ± 81	1,339 ± 89
INO-4885	1,363 ± 125	1,688 ± 179	1,542 ± 175	1,656 ± 195	1,425 ± 104	1,314 ± 89
HR, beats/min
Sham	98 ± 3	103 ± 6	100 ± 5	101 ± 5	106 ± 7	102 ± 3
Control	96 ± 5	136 ± 11*	128 ± 8*	141 ± 10*	145 ± 11*	153 ± 7*
INO-4885	97 ± 1	120 ± 8	104 ± 7*†	104 ± 5*†	112 ± 4*†	119 ± 4*†
CI, l·min−1·m−2
Sham	6.5 ± 0.1	6.9 ± 0.2	6.7 ± 0.3	6.5 ± 0.2	6.1 ± 0.3	6.2 ± 0.2
Control	6.1 ± 0.4	5.9 ± 0.6	6.0 ± 0.4	5.9 ± 0.4	5.7 ± 0.6	5.4 ± 0.5
INO-4885	5.8 ± 0.4	5.8 ± 0.3	5.8 ± 0.5	5.4 ± 0.5	6.0 ± 0.4	6.3 ± 0.4
LAP, mmHg
Sham	9 ± 1	11 ± 1	11 ± 1	11 ± 1	11 ± 1	10 ± 1
Control	9 ± 1	8 ± 1	10 ± 1	9 ± 1	10 ± 1	11 ± 1
INO-4885	8 ± 1	9 ± 1	12 ± 1	11 ± 1	11 ± 1	10 ± 1
SVI, ml·beat−1·m−2
Sham	67 ± 2	68 ± 6	65 ± 5	65 ± 2	59 ± 4	61 ± 2
Control	64 ± 2	46 ± 6*	48 ± 5*	42 ± 3*	39 ± 3*	36 ± 3*
INO-4885	60 ± 4	42 ± 3*	56 ± 4*	51 ± 3*	54 ± 5†	53 ± 4†
LVSWI, g·m−1·m−2
Sham	85 ± 3	93 ± 6	93 ± 6	89 ± 5	73 ± 5	77 ± 3
Control	76 ± 3	62 ± 8*	66 ± 6*	57 ± 5*	48 ± 4*	43 ± 5*
INO-4885	76 ± 3	58 ± 3*	79 ± 3	72 ± 4*	75 ± 8†	73 ± 6†
MPAP, mmHg
Sham	20 ± 1	25 ± 2	26 ± 0	25 ± 0	24 ± 0	24 ± 1
Control	20 ± 1	26 ± 1	27 ± 1	30 ± 2*	28 ± 1	30 ± 2*
INO-4885	21 ± 0	24 ± 1	25 ± 1	28 ± 2	27 ± 1	29 ± 2*
PVRI, dyn·s·cm−5·m−2
Sham	102 ± 5	106 ± 7	135 ± 11	118 ± 10	131 ± 9	116 ± 8
Control	115 ± 13	149 ± 11*	158 ± 14	201 ± 17*	170 ± 26	193 ± 18*
INO-4885	140 ± 10	145 ± 20*	142 ± 21	185 ± 22*	159 ± 20	170 ± 21*

Values are means ± SE. MAP, mean arterial pressure; SVRI, systemic vascular resistance index; HR, heart rate; CI, cardiac index; LAP, left atrial pressure; SVI, stroke volume index; LVSWI, left ventricular stroke work index; MPAP, mean pulmonary arterial pressure; PVRI, pulmonary vascular resistance index.

^*P < 0.05 vs. sham.

^†P < 0.05, INO-4885 vs. control.

NOx levels and MPO activity.

NOx levels in the plasma and lung lymph were similarly increased toward sham after 12 and 24 h in both injured groups. Treatment had little or no effect on this variable (Fig. 4). MPO activity in lung tissue, an index of neutrophil accumulation, was similarly increased in control and treated animals (2.2 ± 0.2, 4.2 ± 0.7, and 3.6 ± 0.5 U/g dry tissue in sham, control, and INO-4885 groups, respectively; P < 0.05, sham vs. control and INO-4885).

Fig. 4.

Impact of ONOO⁻ decomposition catalyst INO-4885 on plasma (A) and lung (B) lymph concentrations of nitrite/nitrate (NOx) in sheep with combined burn and smoke inhalation injury. Values are means ± SE. †P < 0.05 vs. sham.

IL-8, 3-NT, PAR, and VEGF levels.

The injury-related increases in IL-8, 3-NT, and PAR protein expressions were significantly attenuated in the treatment group. Protein expression of VEGF was similarly increased in the control and INO-4885 groups (Fig. 5).

Fig. 5.

Impact of ONOO⁻ decomposition catalyst INO-4885 on IL-8 (A), poly(ADP-ribose) (PAR, B), 3-nitrotyrosine (3-NT, C), and VEGF (D) protein expressions measured by immunoblotting in lung homogenates of sheep with combined burn and smoke inhalation injury. Each group includes 5 animals. Values are means ± SE. †P < 0.05 vs. sham. *P < 0.05, INO-4885 vs. control.

Immunohistochemical staining of PAR in the lungs.

After burn and smoke inhalation injury, PAR was markedly increased in lung tissue of untreated control animals vs. sham-injured animals (Fig. 6A). This increase in PAR was prevented in the INO-4885 group. The semiquantitative PAR positivity scores of small airways (Fig. 6B) and pulmonary glands (Fig. 6C) were significantly increased vs. sham in the control group, but not in the INO-4885 group.

Fig. 6.

Impact of ONOO⁻ decomposition catalyst INO-4885 on PAR polymerase activity in lung tissue of sheep with combined burn and smoke inhalation injury. PAR was stained with a monoclonal antibody (A), and immunohistochemistry stain slide was semiquantified and scored for intensity of PAR staining in small airways (B) and pulmonary glands (C). Each group includes 5 animals. Values are means ± SE. †P < 0.05 vs. sham. *P < 0.05, INO-4885 vs. control.

DISCUSSION

The key findings of the study are that administration of a potent ONOO⁻ decomposition catalyst in sheep subjected to combined burn and smoke inhalation injury reduced pulmonary microvascular hyperpermeability to fluids and proteins and attenuated the injury-induced deterioration of pulmonary function. The treatment was further associated with markedly reduced expressions of IL-8, PAR, and 3-NT in lung tissue.

The current findings complement our previous studies and the overall hypothesis based on the findings that PARP activates NF-κB (9). The chemicals in toxic smoke stimulate the release of neuropeptides, such as calcitonin gene-related peptide, which then activate NO synthase (NOS) to cause the release of RNS (17, 24). RNS, in turn, damage DNA to cause the activation of PARP (24). PARP activates NF-κB (9), which upregulates IL-8 and inducible NOS, setting into motion a positive-feedback loop. We have confirmed much of this hypothesis by blocking calcitonin gene-related peptide (17), different isoforms of NOS (5, 24), PARP (25), and now RNS.

We previously demonstrated that 3-NT, as an indirect measure of ONOO⁻ formation, is markedly elevated in lung tissue of sheep subjected to combined burn and smoke inhalation injury (6, 24, 28, 36). Formation of ONOO⁻ may influence the inflammatory response to this injury at multiple levels. It has been shown that ONOO⁻ stimulates IL-8 gene expression and IL-8 production of human leukocytes (37). In addition, high levels of ONOO⁻ may induce cell death, in part through activation of the PARP pathway secondary to DNA damage. Activation of PARP, in turn, may lead to the depletion of cellular NAD⁺ and ATP stores, which results in the impairment of redox regulation and energy depletion with subsequent necrotic cell death (11, 34). The current study demonstrates that administration of an ONOO⁻ decomposition catalyst not only reduced the degree of ONOO⁻ formation, as measured by 3-NT, but also limited the protein expressions of IL-8 and PAR in lung tissue. These findings confirm that increased PAR formation is a downstream action of ONOO⁻. Reduced formation of PAR in lung tissue has been confirmed by immunohistochemical staining and Western blot techniques.

Previous experiments using the same model of burn- and smoke inhalation-induced lung injury demonstrated that excessively produced NO significantly contributed to the severity of the disease. The blockade of NOS limited the degree of lung injury (5, 28). NO reacts with superoxide, yielding ROS and RNS, such as ONOO⁻. Formation of ONOO⁻, in turn, may induce lung tissue damage by oxidizing and nitrating/nitrosating proteins and lipids (29, 30) or activating PARP (11, 34). However, it is well known that NO plays multiple physiological regulatory roles. Complete blockade of NO production for the purpose of ONOO⁻ formation prevention may therefore cause significant adverse effects, outweighing the advantages of limited ONOO⁻ formation. Thus the downstream blockade of the NO-ONOO⁻ pathway appears to be advantageous. Notably, plasma and lymph concentrations of stable NO metabolites were not affected by administration of INO-4885 in the current study, suggesting that the treatment did not inhibit the expression and activation of NOS, respectively. The beneficial pulmonary effects of catalytic ONOO⁻ decomposition in this study were similar to the effects of NOS inhibition in previous experiments (5, 24, 28, 36). Hence, the results of the current study suggest that downstream actions of inducible NOS-dependent ONOO⁻ explain most effects previously attributed to NO.

Increased formation of ONOO⁻ has further been shown to induce the expression of VEGF by activating signal transducer and activator of transcription 3 (22). VEGF is one of the most potent known natural permeability factors (3, 7). It has been demonstrated that overexpression of VEGF contributes to pulmonary vascular hyperpermeability and lung edema formation (14, 15). Furthermore, there is evidence that VEGF is involved in the development of vascular hyperpermeability in patients with acute respiratory distress syndrome (32). In the current study, induction of acute lung injury by burn and smoke inhalation injury was associated with marked increases in transvascular fluid flux, as evidenced by significant elevations in Q̇_L and lung wet-to-dry weight ratio. These markers of vascular hyperpermeability and lung edema formation were significantly attenuated in the treatment group, indicating that ONOO⁻ plays a crucial role in this process. However, VEGF expression in lung tissue was similarly enhanced in both injured groups. Thus we conclude that, in the current experimental system, VEGF expression is induced by mechanisms other than ONOO⁻ formation. It should be pointed out, nevertheless, that VEGF expression has only been measured at one time point in the lung, i.e., after completion of the study. We cannot exclude the possibility that VEGF expression in the lung could have been significantly reduced at earlier time points.

Previous studies in the same animal model have demonstrated that combined burn and smoke inhalation injury induces significant airway obstruction, which is associated with reduced ventilation or atelectasis of well-perfused regions of the lung (2, 35). This situation frequently requires the elevation of ventilatory pressures and leads to a ventilation-perfusion mismatch, as evidenced by the elevation of Qs/Qt (5, 35). Burn- and smoke inhalation-induced airway obstruction is caused by several pathophysiological mechanisms. Increased pulmonary microvascular permeability represents one major contributor to airway narrowing and the development of obstructive casts by fostering airway wall edema formation and the leakage of procoagulant factors containing exudates into the airways (2, 4). The attenuation of pulmonary vascular hyperpermeability by INO-4885 in the current study, therefore, most likely accounts for the observed reduction of ventilatory pressures and pulmonary shunting in the treatment group.

While the injury-related deteriorations of renal and hepatic function were not affected by administration of the ONOO⁻ decomposition catalyst, there was some evidence of improved myocardial performance. Calculated stroke volume index and left ventricular stroke work index were significantly higher in treated than untreated control animals, suggesting improved myocardial contractility by the neutralization of ONOO⁻. The cardiac index was similar between groups, obviously because of compensation for the reduced contractility by a marked increase in heart rate in the control group. However, the current study was designed to evaluate the pulmonary effects of ONOO⁻ neutralization; thus the mechanism of possible effects on cardiac performance has not been explicitly assessed.

Consistent with previous studies, there was indirect evidence of extrapulmonary vascular hyperpermeability following the double-hit injury (17). In the control group, C_P and π_P were significantly reduced. The hypoproteinemia was unlikely to be a dilutional phenomenon, because there was a tendency for hematocrit and hemoglobin to rise as fluid was administered. The combination of positive net fluid balance and hypoproteinemia in untreated control sheep was probably the result of a transvascular leakage of fluids and proteins from the systemic circulation into the interstitium. In the treated group, the decreases in C_P and π_P were significantly less than in controls. Experiments are needed to address whether the administration of the ONOO⁻ decomposition catalyst has reduced VEGF expressions in extrapulmonary organs and, thus, may have contributed to the attenuation of extrapulmonary vascular hyperpermeability and the extent to which it occurred.

Some of the limitations of the current study are as follows. 1) No dose response was conducted, because the costs of large animal studies make such experiments prohibitively expensive. Nevertheless, the dose selected here was based on previous dose-response studies and internal pilot studies and clearly represents a highly effective dose. 2) We measured 3-NT expression as a marker of ONOO⁻ formation. However, 3-NT is not an ONOO⁻-specific marker, because it may be the result of processes other than ONOO⁻ production (1). Although INO-4885 has been suggested to be a selective and potent neutralizer of ONOO⁻ (12), similar to most studies using pharmacological tools, one cannot completely exclude the possibility that the compound also affects some other radical and oxidant pathways in vivo. 3) The study design included a fixed end point to harvest comparable lung tissue samples. Consequently, it was not feasible to obtain survival data. 4) In the present study, only combined burn and smoke inhalation injury (not the individual components of the injury) has been studied. However, we previously demonstrated that no significant lung injury occurs with burn alone (27), but when smoke inhalation is combined with burn injury, the physiological responses of the lung are different and more severe than with either injury alone (19, 26). Because most patients with inhalation injury also suffer from severe burns, we chose this model to closely imitate this clinical situation.

In conclusion, the current study demonstrates beneficial pulmonary effects of an ONOO⁻ decomposition catalyst in sheep subjected to combined burn and smoke inhalation injury. The compound significantly attenuated the deteriorations in pulmonary oxygenation and shunting, as well as markers of transvascular fluid flux and edema formation. The beneficial effects of INO-4885 may, in part, be attributed to reduced PAR formation in the lung. In summary, catalytic ONOO⁻ decomposition may represent a useful therapeutic adjunct for patients with these injuries.

GRANTS

This study was supported by National Institute of General Medical Sciences Grants P012 GM-066312, R01 GM-060688, and GM-60915 and Shriners Burns Institute Grants 85041, 84050, and 84080.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.