ORIGINAL ARTICLE

Group VIB Ca2+-independent phospholipase A2F is associated with acute lung injury following trauma and hemorrhagic shock Koji Morishita, MD, PhD, Junichi Aiboshi, MD, Tetsuyuki Kobayashi, PhD, Yuri Yokoyama, MS, Saori Mikami, MD, Jiro Kumagai, MD, Keiko Onisawa, BS, and Yasuhiro Otomo, MD, PhD, Tokyo, Japan

BACKGROUND: Gut-derived mediators are carried via mesenteric lymph duct into systemic circulation after trauma/hemorrhagic shock (T/HS), thus leading to acute lung injury (ALI)/multiple-organ dysfunction syndrome. Phospholipase A2 (PLA2) is a key enzyme for the production of lipid mediators in posthemorrhagic shock mesenteric lymph (PHSML). However, the precise functions of PLA2 subtype, such as cytosolic PLA2, secretory PLA2, and Ca2+-independent PLA2, in the acute phase of inflammation have remained unclear. Our previous study has suggested that the activation of Group VIB Ca2+-independent PLA2F (iPLA2F) may be associated with increased lyso-phosphatidylcholines (LPCs) in the PHSML. Therefore, our purpose was to verify the role of iPLA2F on the production of 2-polyunsaturated LPC species and the pathogenesis of T/HS-induced ALI using an iPLA2F-specific inhibitor, R-(E)-6(bromoethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (R-BEL). METHODS: Male Sprague-Dawley rats were anesthetized and cannulated in blood vessels and mesenteric lymph duct. Animals in the T/HS group underwent a midline laparotomy plus hemorrhagic shock (mean arterial pressure, 35 mm Hg, 30 minutes) and 2-hour resuscitation with shed blood and 2 normal saline. Trauma/sham shock rats were performed the identical procedure without hemorrhage. R-BEL or DMSO was administered 30 minutes before T/HS or trauma/sham shock. Polyunsaturated LPCs and arachidonic acid in the PHSML were analyzed with a liquid chromatography/electrospray ionizationYmass spectrometry. Furthermore, ALI was assessed by lung vascular permeability, myeloperoxidase activity, and histology. RESULTS: T/HS increased 2-polyunsaturated LPCs and arachidonic acid in the PHSML. The R-BEL pretreatment significantly decreased these lipids and also inhibited ALI. CONCLUSION: The iPLA2F enzyme is possibly involved in the pathogenesis of ALI following T/HS through the mesenteric lymph pathway. (J Trauma Acute Care Surg. 2013;75: 767Y774. Copyright * 2013 by Lippincott Williams & Wilkins) KEY WORDS: Group VIB Ca2+-independent phospholipase A2F; mesenteric lymph; hemorrhagic shock; acute lung injury.

T

he gut-lymph organ failure hypothesis is supported by in vivo studies showing that the ligation of mesenteric lymph duct prevents acute lung injury (ALI) after hemorrhagic shock in rodents,1 and the cross-transfusion of posthemorrhagic shock mesenteric lymph (PHSML) into naive rat leads to polymorphonuclear leukocyte (PMN) accumulation and provokes lung tissue damage.2 Gonzalez et al.3 showed that the nonionic lipid fraction extracted from PHSML primes PMN for increased superoxide (O2j) production, increases adhesion molecule surface expression, and also inhibits PMN apoptosis. Taken together, mesenteric lymph is central in the pathogenesis of ALI/multiple-organ dysfunction syndrome (MODS) following trauma/hemorrhagic shock (T/HS), but the fundamental mechanism still remains ill defined.4

Phospholipase A2 (PLA2)Ymediated lipid mediators in PHSML is involved in the inflammatory process of T/HSinduced ALI.5 However, the class of PLA2, cytosolic PLA2 (cPLA2), secretory PLA2 (sPLA2), and Ca2+-independent PLA2 (iPLA2), which is responsible for the lipid production, is not identified yet.6 Our previous study has shown that PHSML contains increased lyso-phosphatidylcholine (LPC) species,7 which are possibly produced by the PLA1 function of Group VIB Ca2+-independent PLA2F (iPLA2F) possessing both PLA1 and PLA2 activities.8 These lipid mediators are biologically active for PMN cytotoxicity. The aim of this study was therefore to verify the role of iPLA2F on the production of 2-polyunsaturated LPC species and the pathogenesis of T/HS-induced ALI using R(E)-6-(bromoethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran2-one (R-BEL), an iPLA2F-specific inhibitor.

Submitted: December 29, 2012, Revised: July 31, 2013, Accepted: July 31, 2013. From the Departments of Acute Critical Care and Disaster Medicine (K.M., S.M., Y.O.) and Molecular Pathophysiology (J.K.), Graduate School of Medical and Dental Sciences, and Shock, Trauma and Emergency Medical Center (J.A.), Tokyo Medical and Dental University; and Department of Biology (T.K., Y.Y., K.O.), Faculty of Science, Ochanomizu University, Tokyo, Japan. This study was presented as a poster at the 70th Annual Meeting of the American Association for the Surgery of Trauma and Clinical Congress of Acute Care Surgery, September 2011, Chicago, Illinois. Address for reprints: Junichi Aiboshi, MD, Shock, Trauma and Emergency Medical Center, Tokyo Medical and Dental University, Tokyo, Japan, 1-5-45 Yushima, Bunkyo-ku, Tokyo, Japan, 113-8510; email: [email protected].

MATERIALS AND METHODS

DOI: 10.1097/TA.0b013e3182a924f2

The experimental protocols were approved by the Animal Care Committee of the Tokyo Medical and Dental University School of Medicine in accordance with our institutional guidelines. Male Sprague-Dawley rats were housed in cages in an environmentally controlled room at a temperature of 23-C (1-C) with a 12-hour light/12-hour dark cycle and allowed standard laboratory chow and tap water ad libitum. Animals (353 [37] g) were intraperitoneally anesthetized with ketamine (50 mg/kg) and xylazine (10 mg/kg) before all procedures.

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Materials All materials were purchased from Sigma Chemical (St. Louis, MO) unless specified otherwise. Ketamine was purchased from Daiichi Sankyo Co., Ltd. (Tokyo, Japan), and polyethylene tubes (Sp45: ID, 0.58 mm; OD, 0.96 mm) were obtained from Natsume Seisakusho Co., Ltd. (Tokyo, Japan). Heparin was purchased from Mochida Pharmaceutical Co., Ltd. (Tokyo, Japan). R-BEL was purchased from Cayman Chemical Co., Ltd. (Ann Arbor, MI).

T/HS Model The rats were anesthetized, and then, the left femoral artery and right jugular vein were cannulated with a polyethylene tube. A right medial visceral rotation was performed through a midline celiotomy (trauma) to expose the mesenteric lymphatic duct, which was cannulated. The tubing was exteriorized through the wound in the midline, and then the abdomen was closed. Nonlethal hemorrhagic shock was induced via the jugular vein cannulation until mean arterial pressure (MAP) reduced to 35 mm Hg and maintained for 30 minutes. At the end of shock period, animals in the T/HS group were resuscitated during 2 hours with shed blood plus 2 normal saline. MAP was continuously monitored through the femoral arterial pressure with a LabChart (AD Instrument Pty Ltd, New South Wales, Australia). The trauma/sham shock (T/SS) animals underwent the identical anesthesia and surgical procedure without hemorrhage. Mesenteric lymph was collected on ice during the hemorrhagic shock phase and the fluid resuscitation phase that was divided into the first hour (R1 phase) and the second hour (R2 phase) by definition. Lymph sample from each group was centrifuged at 13,000 G for 5 minutes to remove the cellular component, and the supernatant was stored at j80-C. An additional experiment was performed to evaluate the effect of an iPLA2F-specific inhibitor on ALI following T/HS. Rats underwent the identical anesthesia and surgical procedure without mesenteric lymph duct drainage. The lung injury was assessed by vascular permeability, myeloperoxidase (MPO) activity, and histology. After thoracotomy was performed at the end of the resuscitation, the lungs were perfused free of blood with normal saline and then blotted dry and weighed. The level of lactate in arterial blood was measured at the end of the shock and resuscitation with a blood gas analyzer (ABL800 FLEX, Radiometer. Co., Ltd., Copenhagen, Denmark).

Experimental Design The rats were randomly allocated into four groups as follows: T/SS (n Q 4) or T/HS (n Q 4) pretreated with the intravascular injection of R-BEL or DMSO 30 minutes before the initiation of T/HS. The final plasma concentrations of R-BEL and DMSO were 10 KM and 0.02%, respectively.

Ca2+-Independent PLA2F Inhibitor BEL, an inhibitor selective for iPLA2, is useful for elucidating the role of this enzyme in various settings. It has 100fold selectivity for iPLA2 in comparison with cPLA2 and sPLA2 isoforms.9 Enantiomers, R-BEL and S-BEL, further allow discrimination between the major isoforms of iPLA2, since they are 10-fold selective for iPLA2F and iPLA2A, respectively.10 R-BEL is an irreversible, mechanism-based inhibitor 768

of iPLA2F.11 Treatment with 5-KM R-BEL yields more than 80% inhibition of iPLA2F activity.8 R-BEL does not completely inhibit iPLA2A except at high doses (20Y30 KM).10 The IC50 for iPLA2F was approximately 1 uM.8.8 Based on the previous information, the 10-KM R-BEL was selected as an optimal concentration in this study.

Mesenteric Lymph Lipid Extraction The lipids present in the PHSML of the R2 phase were extracted using the method of Bligh and Dyer.12 Specifically, 100 KL of the PHSML was used for lipid extraction. The mesenteric lymph was diluted with chloroform, and methanol was added to perform a chloroform-methanol-aqueous (1:2:0.8, vol/vol) extraction at 4-C for 30 minutes with stirring. Chloroform and 10-mM Tris-HCl (pH 7.4) were added to perform a chloroform-methanol-aqueous (1:1:0.9, vol/vol) extraction. The mixtures were stirred vigorously and centrifuged at 1,400 G for 10 minutes at 4-C. The upper phase was extracted once more with chloroform, and the lower phases were combined and dried under a nitrogen gas stream. The lipids were dissolved in chloroform-methanol (2:1, vol/vol) and stored at j80-C.

LC/ESI-MS/MS Analysis of LPC Species 1-heptadecanoyl-2-hydroxy-sn-glycero-3phosphocholine (C17:0-LPC) was added to provide the internal standard. The lipids were separated by liquid chromatography separation using an Intertsil SIL-100A column (GL Science, Tokyo, Japan). The mobile phase was acetonitrilemethanol-water 18:11:1, vol/vol/vol plus 0.1% ammonium formate, pH 6.8 at a flow rate of 200 KL/min. Electrospray ionizationYmass spectrometry analyses were performed using a Q TRAP triple quadruple-linear ion trap mass spectrometer (AB SCIEX, Foster, CA) with an HP1100 HPLC system (Agilent Technology, Santa Clara, CA). The source parameters were curtain gas of 40.00 psi, ion spray voltage of 5.00 kV, collision gas pressure of 5.00, ion source gas of 70.00 psi, interface heater of 500-C. The analyses were performed in the positive ion mode using multiple-reaction monitoring analyses to determine LPC molecular species using the specific transitions as follows: the mass/charge (m/z) for their molecular anion M- and their specific daughter ion, m/z 496.2 Y 184.2 for (C16:0) LPC; m/z 510.2 Y 184.2 for heptadecanoyl (C17:0) LPC; m/z 520.2 Y 184.2 for linoleoyl (C18:2) LPC; m/z 522.2 Y184.2 for oleoyl (C18:1) LPC; m/z 524.2 Y 184.2 for stearoyl (C18:0) LPC; m/z 544.2 Y 184.2 for arachidonoyl (C20:4) LPC; m/z 568.2 Y 184.2 for docosahexaenoyl (C22:6) LPC. The lipids in the PHSML were delivered to systemic circulation via mesenteric lymph duct. The flow volume of mesenteric lymph from the R2 phase of the T/HS group consistently increases almost fourfold in comparison with that of the T/SS group owing to the increased vascular permeability after T/HS (Table 2). Therefore, the intensities of these lipids in mesenteric lymph were adjusted by the volume of mesenteric lymph collected during the R2 phase. In addition, the LPC positional isomers, 1-acyl and 2-acyl LPCs, were analyzed to reveal the effect of R-BEL. The relative intensity (%) of each * 2013 Lippincott Williams & Wilkins

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TABLE 1. Histology Grading Scale Grading Score

Inflammatory cells per high-power field Interstitial edema Alveolar hemorrhage Pulmonary edema Alveolar integrity

0

1

2

3

4

G5

6Y10

11Y15

16Y20

920

None/normal None/normal G5% of alveoli Normal

Minimal/moderate Minimal/moderate 5Y25% alveoli Abnormal

Severe Severe 925% alveoli V

V V V V

V V V V

LPC molecular species was expressed compared with the intensity of internal standard (C17:0-LPC) of 1-acyl isomer.

LC/ESI-MS/MS Analysis of Arachidonic Acid D8-AA was added as the internal standard. Solvent A (8.3-mM acetic acid (pH 7.4)) and solvent B (AcCN/MeOH; 65/35; vol/vol) were used. The extracted lipids were injected into a HPLC system using C18 column (Intertsil, GL Science, Tokyo, Japan) and eluted at a flow rate of 200 KL/min, with a linear gradient from 45% to 98% of mobile phase B. Solvent B was increased from 45% to 75% in 13 minutes to 98% in 2 minutes and held at 98% for another 11 minutes. The HPLC system (HP1200HPLC, Agilent Technologies) was directly interfaced into the electrospray source of a Q TRAP triple quadruple mass spectrometer where analyses were performed in the negative ion mode using multiple-reaction monitoring of the specific transitions:13 m/z 303.5 Y 259.2 for arachidonic acid (AA), m/z 311.3 Y 267.2 for d8-AA. The source parameters were as follows: curtain gas of 20.00 psi, ion spray voltage of 4.50 kV, collision gas pressure of 18.00, nebulizer gas of 40.00 psi, and turbo gas of 10.00 psi. The intensity of AA was also adjusted based on the lymph volume of the R2 phase.

Lung Vascular Permeability Lung vascular permeability was measured at the end of the resuscitation phase using the Evans blue dye (EBD) technique as previously described.14 Briefly, EBD (30 mg/kg) was intravascularly injected 1 hour before termination of the experiment followed by harvesting the lungs at the end of resuscitation. Samples were homogenized in formamide. Homogenized tissue was incubated for 24 hours at 37-C and then centrifuged at 10,000 G for 20 minutes. The optical density of the supernatant was determined at 620 nm with a spectrometry (VERSA Max, Molecular Devices Inc., Sunnyvale, CA). The concentration of extravasated EBD (microgram per gram of tissue weight) in lung homogenates was calculated against a standard curve (R2 = 0.99).

MPO Assay The accumulation of PMN in the lung was evaluated using the MPO assay. Briefly, lung tissue was homogenized in phosphate-buffered saline in an ice bath. The homogenized tissue was stored at j80-C to lyse cells. The thawed sample was centrifuged for 30 minutes at 12,000 G at 4-C. MPO in the supernatant was measured following the instruction of MPO assay kit (Northwest Life Science Specialties, Vancouver, WA). The MPO activity (unit of MPO per milligram of tissue weight) was measured at 412 nm with a spectrometry.

Lung Histology and Grading The lungs harvested from rats were immediately fixed in 10% buffered formalin (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and the tissue blocks were embedded in paraffin using the conventional methods. The 4-Km-thick sections from those blocks were stained by hematoxylin and eosin. The slides were observed under a standard light microscope, and lung injury was scored according to the histology grading scale15 as outlined in Table 1.

Statistical Analysis The results are expressed as the mean (SD). An analysis of variance with the post hoc Scheffe multiple comparison test was used for comparisons among the four groups. Analysis of lung injury grading was performed using Kruskal-Wallis nonparametric analysis of variance test. p G 0.05 was considered to be statistically significant.

RESULTS Evaluation of Shock and Resuscitation Status The values of lactate in the DMSO + T/HS and R-BEL + T/HS groups were not significantly different at the end of the shock and resuscitation. The lymph flow during the R2 phase in the DMSO + T/HS and R-BEL + T/HS groups was similar (Table 2).

LC/ESI-MS/MS of LPC Species Each intensity of polyunsaturated (C18:2, C20:4, C22:6, C18:1) LPC species of the DMSO + T/HS group significantly increased compared with that of the DMSO + T/SS group, whereas the pretreatment with R-BEL decreased that of polyunsaturated LPC species (n = 5, p G 0.05) (Fig. 1A). Similarly, the analysis of 2-acyl LPC species revealed that each intensity of 2-polyunsaturated (C18:2, C20:4, C22:6, C18:1) LPC species of the DMSO + T/HS group significantly increased in comparison with that of the DMSO + T/SS group and decreased to the level of DMSO + T/SS by the R-BEL administration (n = 5, p G 0.05) (Fig. 1C). When comparing 1-acyl LPC species with 2-acyl LPC species, each intensity of 2-polyunsaturated (C18:2, C20:4) LPC species of the DMSO + T/HS group markedly increased more than that of 1-polyunsaturated LPC species of the DMSO + T/HS group (Fig. 1B and C).

LC/ESI-MS/MS of AA The intensity of the DMSO + T/HS group (409.0% [42.0%]) significantly increased in comparison with that of

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TABLE 2. Physiologic Parameters Lactate, mmol/L MAP, mm Hg Lymph flow, HL/h

Postshock Postresuscitation Start End R2 phase

DMSO + T/SS

R-BEL + T/SS

DMSO + T/HS

R-BEL + T/HS

1.13 (0.51)* 1.64 (0.31) 84.3 (10.2) 91.7 (11.3) 485.7 (156.0)*

1.10 (0.45)* 1.53 (0.53) 87.0 (12.7) 90.4 (13.9) 514.2 (174.9)*

6.43 (0.73)** 1.53 (0.50) 88.6 (10.8) 87.3 (8.8) 2,428.8 (826.2)**

6.07 (0.81)** 1.65 (0.57) 84.2 (12.4) 85.0 (9.0) 2,090.0 (521.3)**

MAP was continuously monitored through the femoral artery and evaluated at the start/end of the experiment. The level of lactate was measured at the end of shock and resuscitation with a blood gas analyzer. Mesenteric lymph was collected during the second hour of resuscitation. Data are expressed as mean (SD) (each group n Q 4). * and ** indicate that values with different symbols are significantly different (p G 0.05).

Figure 1. Mass spectrometry of lysophosphatidylcholine (LPC). Lipids in the PHSML collected during the R2 phase were extracted, and each LPC molecular species was evaluated using mass spectrometry. Considering the delivery of these lipids in the PHSML to systemic circulation, the intensity of each LPC molecular species was adjusted by the lymph flow volume of the R2 phase (A). LPC positional isomers, 1-acyl LPCs and 2-acyl LPCs, were also analyzed (B and C). Data (%) were expressed as the mean (SD) (n = 5). *Significant difference (p G 0.05). 770

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Figure 2. Mass spectrometry of AA. AA in the PHSML collected during the R2 phase were extracted and then evaluated using mass spectrometry. Considering the delivery of AA in the PHSML to systemic circulation, the intensity of AA was adjusted by the lymph flow volume of the R2 phase. Data (%) were expressed as the mean (SD) (n = 4). *, ¶, and † indicate that values within the four bars with distinct symbols are significantly different (p G 0.05).

the DMSO + T/SS group (160.3% [55.4%]) (n = 4, p G 0.05) (Fig. 2). The R-BEL pretreatment showed a significant decrease in the production of AA (293.4% [14.0%]) (n = 4, p G 0.05) (Fig. 2).

Lung Vascular Permeability and MPO Activity Lung vascular permeability and MPO activity of the R-BEL + T/HS group significantly decreased in comparison with those of the DMSO + T/HS group and were similar to those of DMSO + T/SS and R-BEL + T/SS groups (n Q 4, p G 0.05) (Table 3).

Lung Histology and Grading The appearance of the DMSO + T/HS group showed interstitial edema and inflammatory cell infiltrates. The pretreatment with R-BEL abolished T/HS-induced histological changes (Fig. 3AYC) and significantly decreased the lung histology grading score compared with that of the DMSO + T/HS group (n = 5, p G 0.05) (Fig. 3D).

DISCUSSION Gut-derived mediators reach systemic circulation through mesenteric lymphatics after T/HS, thus leading to ALI/MODS.16 The PLA2-mediated lipid mediators in PHSML are paid attention to as the mechanistic link between splanchnic ischemia/ reperfusion (I/R) and remote organ injury.17 PLA2 enzymes catalyze the cleavage of the sn-2 ester bond of glycerophospholipids to yield polyunsaturated fatty acids and lysophospholipids, functioning in cellular lipid metabolism, membrane remodeling, and lipid mediator signaling.18 A number of mammalian PLA2s

have been identified and classified into several families as follows: sPLA2, cPLA2, iPLA2, platelet-activating factor acetylhydrolases, and lysosomal PLA2s. The cPLA2 family is the best known of these enzymes and plays a major role in releasing AA, a precursor of prostaglandins and leukotrienes, from the cellular membrane.19 AACOCF3, a cPLA2-specific inhibitor, significantly reduces the production of thromboxane B2/leukotriene B4 and attenuates I/R injury in an isolated rat lung model.20 The sPLA2 family affects various biologic events by modulating the extracellular phospholipid environment. It has been found in various body fluids from humans with sepsis/adult respiratory distress syndrome and is strongly correlated with the severity and prognosis of these disorders.21 Koike et al.22 reported that a sPLA2-IIA inhibitor (S-5920/LY315920Na) attenuates ALI after gut I/R in the mice. Taken together, cPLA2 and sPLA2 are involved in the pathogenesis of ALI/MODS following sepsis and I/R. With regard to the iPLA2 family, it mainly contributes to membrane homeostasis and energy metabolism.19 Nine members of the iPLA2 family are identified and expressed in a variety of cells, such as vascular endothelial cells,23,24 PMNs,25 smooth muscle cells, myocytes,26,27 macrophages,28 intestinal epithelial cells,29 and lung alveolar cells.30 The iPLA2 enzyme is activated in response to thrombin,23 tryptase,26 lipopolysaccharide (LPS), proinflammatory cytokines,31 and hypoxia.32 In addition, iPLA2 is expressed during the acute phase (2Y24 hours) in a pleurisy model, earlier than cPLA2 and sPLA2, and induces the sequestration of inflammatory cells into inflamed tissue and the production of proinflammatory mediators.33 These major PLA2s may serve as a modulator at inflamed site in the different timing.33 Thus, compelling evidences suggest that it may be a key enzyme in acute inflammation process. The current study showed that the R-BEL pretreatment attenuates T/HS-induced 2-polyunsaturated LPC species in PHSML. In general, 2-polyunsaturated LPC species are produced by hydrolyzing the sn-1-saturated fatty acyl ester of phosphatidylcholine (PLA1 activity). However, PLA1 is unable to catalyze the selective production of 2-polyunsaturated LPC species owing to the substrate specificity. Recently, iPLA2F is reported to possess both PLA1 and PLA2 activities and functions predominantly as the PLA1 enzyme in the presence of diacyl PC substrates containing a sn-2 polyunsaturated acyl group.8 The PLA1 activity of iPLA2F possibly produces 2-polyunsaturated LPCs in the PHSML. In contrast, heparin

TABLE 3. Lung Injury Measured as Concentration of EBD and Unit of MPO Activity Group DMSO + T/SS R-BEL + T/SS DMSO + T/HS R-BEL + T/HS

Lung Permeability, mg/g Tissue

MPO Activity, U/mg Tissue

0.020 (0.002)* 0.021 (0.002)* 0.038 (0.005)** 0.021 (0.002)*

14.47 (0.40)* 15.36 (1.59)* 20.10 (2.31)** 15.82 (2.33)*

Data are expressed as mean (SD) (each group n Q 4). * and ** indicate that values within the four groups with different symbols are significantly different (p G 0.05).

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Figure 3. Lung histology. The harvested lungs were immediately fixed in 10% buffered formalin. The tissue blocks were embedded in paraffin according to the conventional methods and stained by hematoxylin and eosin. These specimens are representatives of DMSO + T/HS (A), DMSO + T/SS (B), and R-BEL + T/HS (C) (400). The lung injury was scored according to the histology grading scale (D). Data were expressed as the mean (SD) (n = 5). *, ¶, and † indicate that values within the four bars with distinct symbols are significantly different (p G 0.05).

used as an anticoagulant for shed blood may confound our result that T/HS increases 2-polyunsaturated LPC species in PHSML. Qin et al.34 demonstrated that heparin-induced lipase activity is responsible for the endothelial cytotoxic properties of mesenteric lymph. To exclude this confounding factor, we performed an additional experiment using Arixtra, fondaparinux sodium, with no lipase activity instead of heparin. 2-polyunsaturated LPC species in PHSML from Arixtra-treated rats subjected to T/HS significantly increased in accordance with our current study (data not shown). Therefore, the heparininduced lipase activity is not associated with increased polyunsaturated LPCs in PHSML. For the generation of AA in PHSML, some direct/indirect mechanisms of iPLA2F-mediated AA production are suggested. Murakami et al.35 reported that the overexpression of iPLA2F in HEK293 cells augments AA production,while HEK293 cells with iPLA2F small interfering RNA significantly reduce AA release. In addition, Kuwata et al.31 showed that the knockdown of iPLA2F by small interfering RNA in rat fibroblastic 3Y cells attenuates the expression of sPLA2-IIA stimulated by cytokines/LPS. R-BEL also suppresses the cytokine-induced sPLA2-IIA expression. Thus, iPLA2Fmediated AA generation is multifactorial. Our result that the R-BEL pretreatment attenuates ALI suggests that 2-polyunsaturated LPC species and AA produced by the iPLA2F activation are cytotoxic for remote organ tissue. LPCs induce the up-regulation of adhesion molecules expression, the production of cytokines, the secretion of O2j, and the activation of nuclear factor JB in leukocytes, endothelial cells, and smooth muscle cells.36 Our previous study also showed that polyunsaturated (C18:2, C20:4) LPCs induce O2j 772

production and elastase release from human PMN.7 As to the biologic activity of AA, Jordan et al.13 have demonstrated that AA stimulates PMN to produce superoxide anion/leukotriene B4 culminating in lung tissue damage. In vitro studies also show that AA activates PMN for respiratory burst, degranulation, up-regulation of CD11b/CD18 adhesion molecules, adhesion, and antimicrobial activity.37 Collectively, it is possible that 2-polyunsaturated LPC species and AA in the PHSML cause multiple-organ injury following T/HS. There are some limitations in the current study. The pretreatment of R-BEL may inhibit the activation of the iPLA2 enzyme expressed not only in the intestine but also in various cells involved in T/HS-induced lung injury, such as vascular endothelial cells and PMNs. Second limitation is the specificity of R-BEL. The R-BEL concentration we used in this experiment inhibits 85% of iPLA2F activity and to some extent iPLA2A activity.10 Third, unmeasured mediators from injured gut also possibly contribute to lung injury. We could not find any difference in gut injury between sham and T/HS groups owing to less invasive shock. However, in our recent experiment using severe hemorrhagic shock model (25Y30 mm Hg  60 minutes), R-BEL significantly decreases the leakage of EBD into an intraluminal space of small intestine (data not shown). The protective effect of R-BEL on the gut may inhibit the production of indeterminate mediators as well as polyunsaturated LPCs and AA. In conclusion, an iPLA2F-specific inhibitor decreased 2-polyunsaturated LPC species and AA in the PHSML and then attenuated ALI following T/HS. The iPLA2F activation is one possible mechanism by which gut I/R contributes to the * 2013 Lippincott Williams & Wilkins

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pathogenesis of ALI/MODS through proinflammatory lipid mediators in the PHSML. Elucidating the mechanistic link between iPLA2F and ALI/MODS could lead to the development of new therapeutic strategies.

14.

15. AUTHORSHIP K.M. performed the operation and sample collection in the animal experiment. T.K., Y. Y., and K.O. performed the data collection and analysis of mass spectrometry. K.M, J.A., J.K. and S.M. performed the analysis of lung injury. K.M., J.A., T.K., and Y.O. drafted the article. All authors read and approved the final article.

16.

17. DISCLOSURE

18.

This study was supported by Grant-in-Aid for Scientific Research (No. 23592666) from Japan Society of the Promotion of Science (to J.A.).

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