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1 5-lipoxygenase deficiency prevents respiratory failure during ventilator-induced lung injury Pietro Caironi, Fumito Ichinose, Rong Liu, Rosemary C. Jones, Kenneth D. Bloch, and Warren M. Zapol ONLINE DATA SUPPLEMENT

2 Caironi et al., Online data supplement, page 2 EXPANDED METHODS: All experiments were performed after approval by the Massachusetts General Hospital Subcommittee on Research Animal Care. The 5LO-deficient mice (B6;129S-Alox 5tm/fun ) [1] and their wild-type control mice (B6129SF2/J) were obtained from the Jackson Laboratory (Bar Harbor, ME) and were maintained in the animal resource facility of the Massachusetts General Hospital. Mouse model of VILI For mechanical ventilation, mice were anesthetized by intraperitoneal injection of ketamine (120 mg/kg) and xylazine (8 mg/kg); then, a tracheostomy and arterial catheterization were performed as previously described [2]. Volume-controlled ventilation (Harvard Apparatus ventilator 687, Holliston, MA) was initiated with a tidal volume (V T ) of 8 ml/kg of body weight and a respiratory rate (RR) of 100 breaths per minute (b/min), at positive end-expiratory pressure (PEEP) of 1.5 cmh 2 O. After hemodynamic stability was obtained, mice were divided to receive two differing types of mechanical ventilation. Low V T (LV T ) ventilation provided a V T equal to 12% of inspiratory capacity (IC), at positive end-expiratory pressure (PEEP) of 1.5 cmh 2 O, respiratory rate (RR) of 100 breaths/min, and inspired oxygen fraction (FiO 2 ) of HV T ventilation provided a V T equal to 43% of IC, at PEEP of 0 cmh 2 O, RR of 90 breaths/min, and FiO 2 of IC was obtained, for each animal, by constructing a pressure-volume (PV) curve of the respiratory system. After commencing mechanical ventilation, both with low tidal volumes (LV T ) and high tidal volumes (HV T ), carbon dioxide (CO 2 ) was added to inspired gases ( inspired CO 2 fraction), to maintain the arterial partial pressure of CO 2 (PaCO 2 ) within a

3 Caironi et al., Online data supplement, page 3 physiological range, and the CO 2 concentration of exhaled gases was monitored. Anesthesia and paralysis were maintained throughout each study by intra-peritoneal injection respectively of ketamine (30 mg/kg body weight per hour) and xylazine (0.2 mg/kg body weight per hour), and pancuronium (2 mg/kg body weight per hour). Body temperature was kept constant ( C) using a warming pad. Mechanical ventilation was ended either after 10 hours or when (after 7 to 10 hours of HV T ventilation) the elastance of the respiratory system at 0.1 ml lung inflation on the PV curve was greater than 45 cmh 2 O/ml, indicating major deterioration of the respiratory system compliance ( long-term mechanical ventilation ). In pilot studies, this upper elastance limit immediately preceded death. In additional studies, mechanical ventilation was interrupted after 6 hours, to study the time course of the lung injury. Construction of pressure-volume curve of the respiratory system Quasi-static pressure-volume (PV) curve of the respiratory system was performed by stepwise lung inflation with 0.1 ml of oxygen to an airway pressure of 30 cmh 2 O, each step lasting approximately 3.5 seconds. To standardize lung volume history, the PV curve was preceded by a recruitment maneuver (lung inflation to 30 cmh 2 O airway pressure for 30 seconds). Inspiratory capacity (IC) was defined as the inflation volume needed to achieve 30 cmh 2 O airway pressure. Inflation lung volume was expressed as a percentage of IC at baseline for each animal. Monitoring during mechanical ventilation For airway pressure monitoring, a pressure transducer (MP-45, Valydine Engineering Corp., Northridge, CA) was connected to the proximal end of the tracheostomy. To exclude, during HV T ventilation, a possible error of airway pressure measurement caused by the resistance of the

4 Caironi et al., Online data supplement, page 4 endotracheal tube, airway pressures were measured in additional mice (n = 3) by inserting a 1.4- Fr high-fidelity pressure catheter (Millar Instruments, Houston, TX) into the trachea just above the carina. The difference between the two airway pressure measurements was less than 5%. Peak airway pressure and total dynamic positive end-expiratory pressure (PEEP) values were obtained at baseline from airway pressure recording. V T measurements were initially derived from the ventilator, through calibrated cylinder displacement. To take into account the compression volume of the ventilator circuit, the actual delivered V T was recorded in additional mice (n = 3, each strain) using a whole-body plethysmograph (Buxco, Sharon, CT). For each experiment, the actual delivered V T was then computed (see Table 1). To monitor respiratory mechanics during the experiment, a PV curve was performed every hour, as described above. Systemic arterial pressure (SAP) was continuously monitored using fluid-filled membrane transducers (CDXpress, Argon Medical, Athens, TX). During mechanical ventilation, mice received 0.01 ml/g/hour Ringer s lactate through the arterial line. Gas exchange analysis At the end of the experiment, arterial blood was obtained from the left carotid artery, and a blood gas analysis was performed. The alveolar-arterial oxygen tension difference (A-aDO 2 ) was computed, assuming a respiratory quotient of 0.8. Lung edema and microvascular permeability assay To assess pulmonary edema formation, the amount of extravascular lung water (EVLW) in lung tissue was computed as described previously [3]. At the end of the experiment, blood samples

5 Caironi et al., Online data supplement, page 5 were analyzed for hemoglobin concentration and dried in a microwave (1 hour) to compute water content. The right lung was removed from the chest cavity, drained of blood, and then weighed. After the addition of 1 ml distilled water, the lung was homogenized. A portion of the homogenate (0.5 ml) was centrifuged (16000 g, for 8 minutes at 4 C) and, after adding 0.05 g lauryl sulfate for clearing, the supernatant was analyzed for hemoglobin concentration. The remaining portion of homogenate was dried in a microwave (1 hour) to assess the dry lung weight. EVLW was expressed as the weight of water in the extra-vascular space divided by the dry lung weight. To estimate pulmonary vascular permeability, BALF supernatant was analyzed for total protein concentration using the Bradford method (Bio-Rad assay kit, Hercules, CA) with bovine serum albumin used as a standard. Histopathologic analysis Mouse lungs were perfusion-fixed with 0.3% paraformaldehyde and 0.1% glutaraldehyde via both the airway and pulmonary artery and embedded in JB-4-resin. Sections (2 µm in thickness) were stained with 0.05% toluidine blue and microscopically examined by an investigator blinded as to the mouse genotype and ventilation protocol. Calculation of left pulmonary vascular resistance After 6 hours of either LV T or HV T ventilation, mice were ventilated in a standard fashion with a V T of 7 ml/kg body weight, RR of 100 breaths/min, FiO 2 1.0, and a thoracotomy was performed. To monitor the pulmonary artery pressure (PAP), a pulmonary artery catheter (PE10) was

6 Caironi et al., Online data supplement, page 6 inserted into the main pulmonary artery by direct puncture, and connected to a fluid-filled membrane pressure transducer (Argon, Maxxim Medical, Clearwater, FL). Left pulmonary artery blood flow (QLPA) was measured with a small-vessel flow probe connected to a flowmeter (T106; Transonic Instruments, Ithaca, NY). While simultaneously measuring QLPA and PAP, the inferior vena cava (IVC) was partially occluded with a circumferential 5-0 silk ligature to transiently reduce the cardiac output until QLPA was decreased by approximately 50%. To calculate left pulmonary vascular resistance (LPVR), the pressure-flow relationship was constructed by plotting approximately 50 consecutive digitized data points of linear part of PAP and QLPA tracings during transient IVC occlusion. The best fit describing the relationship between PAP and QLPA was obtained by linear regression analysis. The slope of this best-fit line represents incremental LPVR during IVC occlusion. The percent increase in LPVR during left mainstem bronchus occlusion (LMBO) was then computed for each animal. Experimental groups Mice of both sexes, with a body weight range of grams were studied. The experimental groups were matched for sex and weight. Group 1 [baseline]: B6129SF2/J wild-type mice (n=10) and 5LO-deficient mice (n=7) were studied immediately after the instrumentation. Group 2 [wild-type mice, long-term ventilation]: B6129SF2/J wild type mice were studied during a period of LV T ventilation for 6 hours (n=5) or 10 hours (n=12), or during a period of HV T ventilation for 6 hours (n=5) or 10 hours (n=15).

7 Caironi et al., Online data supplement, page 7 Group 3 [baseline, HPV measurement]: B6129SF2/J wild-type mice (n=4) were studied immediately after the instrumentation. Group 4 [wild-type mice, HPV measurement]: B6129SF2/J wild-type mice were studied after a period of 6 hours ventilation with LV T (n=8) or HV T (n=8). Group 5 [pulmonary vasoconstrictor effect of angiotensin II]: after a period of 6 hours of mechanical ventilation with LV T (n=3) or HV T (n=4), B6129SF2/J were studied before and during an intravenous infusion of angiotensin II (5 µg/kg/min), as previously described [4]. Group 6 [5LO-/- mice, long term ventilation]: 5LO-deficient mice were studied during a period of LV T ventilation for 6 hours (n=5) or 10 hours (n=8), or during a period of HV T ventilation for 6 hours (n=5) or 10 hours (n=8). Group 7 [5LO -/- mice, HPV measurement]: 5LO-deficient mice were studied after 6 hours of mechanical ventilation with LV T (n=4) or HV T (n=3). Group 8 [MK886-pretreated]: after intraperitoneal injection of MK886 (Calbiochem, San Diego, CA), a FLAP inhibitor (30 mg/kg, 12 hours and again at 30 minutes before commencing mechanical ventilation) [5], B6129SF2/J wild-type mice were studied during a period of LV T ventilation for 6 hours (n=5) or 10 hours (n=9), or during a period of HV T ventilation for 6 hours (n=5) or 10 hours (n=12).

8 Caironi et al., Online data supplement, page 8 Group 9 [MK571-treated]: after intraperitoneal injection of MK571 (Cayman Chemical Company, Ann Arbor, MI), a cyslt-1 receptor antagonist (10 mg/kg, 1 hour before and every 4 hours during the experiment) [6], B6129SF2/J wild-type mice were studied during a period of HV T ventilation for 6 hours (n=5) or 10 hours (n=6). Exclusion criteria To avoid including confounding factors due to the development of metabolic acidosis or shock during long-term mechanical ventilation, animals with an arterial ph (pha) lower than 7.22 at a PaCO 2 lower than 45 mmhg or a pha lower than 7.17 at the end of the experiment were excluded. For LV T ventilation, 3 animals in the 5LO-deficient group and 1 animal in the MK886- pretreated group were excluded. For HV T ventilation, 2 animals in both the wild-type and MK886-pretreated groups, 3 animals in the 5LO-deficient group, and 1 animal in the MK571- treated group were excluded. The survival analysis including all of the excluded animals did not differ from the analysis reported. Statistical analysis Comparisons between survival curves were performed using a log-rank test. A two-way ANOVA was used to test the effect of time and ventilation mode within different experimental groups, and genotype/treatment within the same ventilation mode. When the ANOVA was significant, a post-hoc Bonferroni t-test was used. If not distributed normally, data were analyzed with ANOVA on ranks, and a Newman-Keuls test was used for post-hoc analysis (Sigma Stat 2.03; SPPS, Inc., Chicago, IL). Statistical significance was defined as P<0.05. All data are expressed as mean±sem.

9 Caironi et al., Online data supplement, page 9 REFERENCES: E1. Chen XS, Sheller JR, Johnson EN, Funk CD. Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene. Nature 1994;372: E2. Ichinose F, Zapol WM, Sapirstein A, Ullrich R, Tager AM, Coggins K, Jones R, Bloch KD. Attenuation of hypoxic pulmonary vasoconstriction by endotoxemia requires 5- lipoxygenase in mice. Circ Res 2001;88: E3. Pearce ML, Yamashita J, Beazell J. Measurement of pulmonary edema. Circ Res 1965;16: E4. Ullrich R, Bloch KD, Ichinose F, Steudel W, Zapol WM. Hypoxic pulmonary blood flow redistribution and arterial oxygenation in endotoxin-challenged NOS2-deficient mice. J Clin Invest 1999;104: E5. Voelkel NF, Tuder RM, Wade K, Hoper M, Lepley RA, Goulet JL, Koller BH, Fitzpatrick F. Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest 1996;97: E6. Blain JF, Sirois P. Involvement of LTD(4) in allergic pulmonary inflammation in mice: modulation by cyslt(1) antagonist MK-571. Prostaglandins Leukot Essent Fatty Acids 2000;62:

10 Caironi et al., Online data supplement, page 10 SUPPLEMENTAL DATA: Online Table E1 Hemodynamics measurements at baseline, after 6 hours of mechanical ventilation, and at the end of the experiment from animals subjected to mechanical ventilation either with LV T or HV T. All values were compared between groups and durations of ventilation by two-way ANOVA for repeated measurements. No significant differences were observed. SAP, systemic arterial pressure; HR, heart rate. Online Table E2 Arterial blood gas analysis in wild-type mice, at baseline and during long-term mechanical ventilation. All values were compared between groups by ANOVA with a post-hoc comparison. *P<0.01 vs. baseline. P<0.001 vs. after 6 hours at the same ventilation. P<0.001, P<0.05 vs. LV T ventilation for the same duration. Online Table E3 Hemodynamics measurements before (baseline) left mainstem bronchus occlusion (LMBO), and 5 minutes after LMBO, in wild-type and 5LO-deficient mice. All values at baseline and after LMBO in each strain were compared by ANOVA with a post-hoc comparison. *P<0.05 vs. LV T ventilation at the same genotype. P<0.05 vs. wild-type mice at the same ventilation. P<0.05 vs. baseline of the same parameter. HR, heart rate; SAP, systemic arterial pressure; PAP, pulmonary arterial pressure; QLPA, left pulmonary arterial blood flow.

11 Caironi et al., Online data supplement, page 11 Online Table E4 Total cell population in BALF obtained at baseline, after 6 hours of mechanical ventilation, and at the end of the experiment from animals subjected to long-term mechanical ventilation either with LV T or HV T. All values were compared between groups and durations of ventilation by two-way ANOVA. *P<0.05 vs. baseline of the same cell type. P<0.05 vs. after 6 hours at the same ventilation. P<0.05 vs. LV T ventilation at the same genotype. P<0.05 vs. wild-type mice at the same ventilation. ll P<0.05 vs. 5LO-deficient mice and MK886-pretreated wild-type mice at the same ventilation. PAMs, pulmonary alveolar macrophages; PMNs, polymorphonuclear cells.

12 Caironi et al., Online data supplement, page 12 Online Table E1 Hemodynamics baseline after 6 hours end of experiment SAP HR SAP HR SAP HR n mmhg b/min mmhg b/min mmhg b/min WT LV T ± ± ± ± ± ± 19 WT HV T ± ± ± ± ± 3 508± 15 5LO-/- LV T ± ± ± ± ± ± 29 5LO-/- HV T ± ± ± ± ± ± 21

13 Caironi et al., Online data supplement, page 13 Online Table E2 Arterial blood gas analysis low V T high V T baseline after 6 hrs end of experiment after 6 hrs end of experiment n = 10 n = 5 n = 12 n = 5 n = 15 PaO 2 (mmhg) 244 ± ± ± ± ± 20*,, PaCO 2 (mmhg) 33 ± 3 33 ± 4 31 ± 2 35 ± 2 34 ± 2 PH 7.35 ± ± ± ± ± 0.02 HCO - 3 (mmol/l) 17.7 ± ± ± ± ± 0.6 Hb (g/dl) 13.7 ± ± ± 0.3* 12.7 ± ± 0.3

14 Caironi et al., Online data supplement, page 14 Online Table E3 Hemodynamics during HPV measurement HR SAP PAP QLPA (beats per minute) (mmhg) (mmhg) (µl/min/g) n baseline LMBO baseline LMBO baseline LMBO baseline LMBO LV T WT ± ± ± 4 73 ± 5 14 ± 1 16 ± ± 6 61 ± 5 LV T 5LO-/ ± ± ± 7 57 ± 8 12 ± 1 14 ± 2 87 ± 8 54 ± 6 HV T WT ± ± ± 8 75 ± 8 15 ± 1 16 ± 1 83 ± 7* 75 ± 6 HV T 5LO-/ ± ± ± 8 54 ± 5 16 ± 1 19 ± ± 8*, 74 ± 4

15 Caironi et al., Online data supplement, page 15 Online Table E4 Cell population in BAL fluid low V T high V T baseline 6 hours end of experiment 6 hours end of experiment n PAMs [x 10 3 ] WT 84 ± 6 80 ± 4 73 ± 4 95 ± 8 11 ± 2*,, PMNs [x 10 3 ] WT 2 ± 1 7 ± 2 14 ± 1*, 31 ± 6*, 27 ± 5*, Others [x 10 3 ] WT 1 ± 0 1 ± 0 1 ± 0 6 ± 1*, 2 ± 0 n PAMs [x 10 3 ] 5LO-/- 88 ± 7 77 ± 2 74 ± 6 89 ± 7 73 ± 3 PMNs [x 10 3 ] 5LO-/- 1 ± 0 7 ± 1 13 ± 2* 7 ± 1 18 ± 2*,, Others [x 10 3 ] 5LO-/- 1 ± 0 1 ± 0 1 ± 0 2 ± 0 1 ± 0 n PAMs [x 10 3 ] WT+MK ± 5 83 ± 6 80 ± 4 80 ± 5 76 ± 3 PMNs [x 10 3 ] WT+MK886 1 ± 0 5 ± 1 10 ± 1 6 ± 1 18 ± 4*,,, Others [x 10 3 ] WT+MK886 0 ± 0 1 ± 0 1 ± 0 1 ± 0 2 ± 1 n 5 3 PAMs [x 10 3 ] WT+MK ± 4 71 ± 3 PMNs [x 10 3 ] WT+MK ± 5 ll 56 ± 3,,ll Others [x 10 3 ] WT+MK571 3 ± 1 2 ± 0