Protection against Oxidative Stress-induced Hepatic Injury by Intracellular Type II Platelet-activating Factor Acetylhydrolase by Metabolism of Oxidized Phospholipids in Vivo*

Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2-/-) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2-/- mice, Pafah2-/- mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2-/- mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl4) was injected into mice, Pafah2-/- mice showed a delay in hepatic injury recovery. Moreover, after CCl4 administration, liver levels of the esterified form of 8-iso-PGF2α, a known in vitro substrate of PAF-AH (II), were higher in Pafah2-/- mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F2α and protects tissue from oxidative stress-induced injury.

Oxidative stress has been implicated in a number of human diseases including atherosclerosis, cancer, neurodegenerative disorders, and aging (1)(2)(3)(4). Polyunsaturated fatty acid-containing lipids are recognized as targets of oxidative damage, readily undergoing peroxidation upon exposure to free radicals. Peroxidation of lipids can greatly alter the physicochemical properties of membrane lipid bilayers, resulting in severe cellular dysfunction. In addition, a variety of lipid byproducts evolve from lipid peroxidation, some of which can exert adverse biological effects (5)(6)(7). Therefore, peroxidized and oxidized phospholipids have to be promptly removed in vivo. It is assumed that these chemically modified lipids are preferentially hydrolyzed by cellular phospholipase A 2 that mainly hydrolyzes fatty acyl chains attached to the sn-2 position of phosphoglycerides, the position where polyunsaturated fatty acyl chains are generally attached. One of the candidate enzymes responsible for the release of oxidized fatty acyl chains from the sn-2 position of membrane phospholipids is thought to be platelet-activating factor acetylhydrolase (PAF-AH) 2 (8).
PAF-AHs were originally identified as enzymes that hydrolyze the acetyl group attached to the sn-2 position of PAF (1-Oalkyl-2-acetyl-sn-glycero-3-phosphocholine), which is a potent signaling phospholipid involved in diverse physiological and pathological events such as inflammation, anaphylaxis, reproduction, and fetal development (9). Three types of PAF-AH have been identified in mammals, namely the intracellular types I and II and a plasma type (10,11). PAF-AHs comprise groups VII and VIII of the PLA 2 superfamily of proteins (12,13).
The initial studies on PAF-AH were performed on plasma PAF-AH (10, 14 -16). Plasma PAF-AH is a 45-kDa secretory enzyme (17) that is associated mainly with low density lipoproteins and high density lipoproteins in plasma (18,19). Plasma PAF-AH has marked selectivity for phospholipids with short acyl chains at the sn-2 position; with chains longer than nine carbons there was essentially no measurable activity (20 -22). * This study was supported by grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and by Core Research for Evolutional Science and Technology (CREST) and Precursory Research for Embryonic Science and Technology (PRESTO) of the Japan Science and Technology Corp. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains a supplemental figure. 1 To whom correspondence should be addressed: 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.: 81-3-5841-4720; Fax: 81-3-3818-3173; E-mail: harai@mol.f.u-tokyo.ac.jp.
Interestingly, the suitability of a phospholipid as a substrate for plasma PAF-AH increases if the sn-2 acyl group has an oxidized functionality, such as an aldehydic or carboxylic group (21,23). These unusual sn-2 acyl groups are generated by oxidative cleavage of long-chain polyunsaturated fatty acyl groups.
Recently it has been reported that oxidatively modified phospholipids such as esterified F 2 -isoprostanes (24) and phospholipid hydroperoxides (25) are good substrates for plasma PAF-AH, suggesting that residue length is not the only factor involved in substrate recognition. In animal models recombinant plasma PAF-AH was effective in treating acute pancreatitis (26), asthma (27), and anaphylactic shock (28). Adenovirusmediated gene transfer of plasma PAF-AH into the liver in apolipoprotein E-deficient mice reduced the level of oxidized low density lipoproteins in the blood (29,30). These suppressive effects of plasma PAF-AH are thought to be due to its ability to hydrolyze PAF and PAF-like oxidized phospholipids. Four percent of the Japanese population lack plasma PAF-AH, and such a deficiency or decrease in plasma PAF-AH activity may be associated with severe asthma (31)(32)(33), atopy (32), stroke, and various cardiovascular diseases (34 -37). Intracellular type II PAF-AH (PAF-AH (II)) is a monomeric 40-kDa enzyme. The amino acid sequence of PAF-AH (II) does not show any similarity to any subunit of PAF-AH (I) but significant identity with plasma PAF-AH. PAF-AH (II) is N-myristoylated at the N terminus and is distributed in both the cytosol and membranes like other N-myristoylated proteins (38). PAF-AH (II) exhibits a substrate specificity that is very similar to plasma PAF-AH. PAF-AH (II) can hydrolyze phospholipids with short to medium length sn-2 acyl chains including truncated chains derived from oxidative cleavage of longchain polyunsaturated fatty acyl groups. However, the activity of this enzyme toward phospholipids with two long (14 -18 carbons) fatty acyl chains is negligible (39,40). We and another group have demonstrated that overexpression of PAF-AH (II) suppresses oxidative stress-induced cell death. Moreover, PAF-AH (II) translocates from the cytosol to the membrane during oxidative stress (38,41). These results strongly suggest that PAF-AH (II) functions as an antioxidant phospholipase. Unlike intracellular type I PAF-AH, PAF-AH (II) is conserved in many organisms, including mammals, frog, fishes, nematodes, and even in yeast. We have shown that the Caenorhabditis elegans orthologue of mammalian PAF-AH (II) is expressed in epithelial cells of C. elegans and plays an important role in epithelial morphogenesis (40). Because PAF is not present in C. elegans (42), it is not likely that PAF-AH (II) functions as a PAF-degrading enzyme. In this study we generated PAF-AH (II)-deficient (Pafah2 Ϫ/Ϫ ) mice by targeted disruption to elucidate the physiological and pathological functions of intracellular PAF-AH (II) in mammals.

EXPERIMENTAL PROCEDURES
Generation of Pafah2 Ϫ/Ϫ Mice-Pafah2 genomic clones were isolated from a mouse 129/SvJ genomic library in the Lambda FIXII vector (Stratagene, La Jolla, CA). A replacementtype targeting vector was constructed; the short arm containing a 1.0-kilobase fragment in intron 7 and the long arm containing a 9.2-kilobase SpeI fragment spanning exons 10 -11 were inserted into the XhoI and NotI sites, respectively, of the vector pPolIIshort-neobpA-HSVTK. Exons 8 -9, which include catalytic motif (GXSXV), were replaced by a neomycin-resistant cassette. The targeting vectors were linearized and electroporated into E14Tg2a embryonic stem cells (ES cells) (43). G418resistant colonies were screened for homologous recombinants by PCR. Candidates of homologous recombinants were verified by Southern blot analysis using fragments at the 5Ј-end of the genes, external to the targeting vectors as probes. Chimeric mice were generated by injection of the ES cells into C57BL/6 blastocysts followed by transfer to foster mothers and backcrossed to C57BL/6 mice. Genotypes were determined by PCR and/or Southern blot analysis of the tail DNA samples. All experiments reported here were performed with mice derived from six to eight generations of backcross-breeding to C57BL/6 mice.
Histological and Immunohistochemical Analyses-Mice under anesthesia were perfused with phosphate-buffered saline. Tissues were dissected and fixed overnight in 4% paraformaldehyde/phosphate-buffered saline at 4°C. Paraffin sections (5 m) were prepared and stained with hematoxylin and eosin. For immunohistochemistry, paraffin sections (5 m) were boiled in a microwave oven in 10 mM sodium citrate buffer (pH 6.0) for antigen retrieval. Subsequent immunodetection was performed using the monoclonal antibody TI10 and Vector M.O.M. peroxidase immunodetection kit (Vector Laboratories, Burlingame, CA). The sections were also counterstained with hematoxylin.
Enzyme Assays-Mouse tissue samples were homogenized in SET buffer followed by centrifugation at 100,000 ϫ g for 1 h to obtain the soluble fraction. Mouse blood samples were obtained by retro-orbital bleeding. Plasma was prepared by centrifugation of the blood at 1800 ϫ g for 10 min. Peritoneal exudate macrophages were suspended in SET buffer and disrupted by sonication for 3 periods of 4 s at 10-s intervals using a Branson Sonifer. The soluble fraction was prepared by centrifugation at 1000 ϫ g for 20 min. PAF-AH activity of the tissue soluble fraction and the plasma were measured as previously described (45).
Oxidized phospholipid-hydrolyzing activity was measured as follows. The standard incubation system for assay comprised 50 mM Tris-HCl (pH 7.4), 5 mM EDTA, 10 nmol of 1-palmitoyl, 2-azeraoyl phosphatidylcholine (PC), and the sample in a total volume of 0.125 ml. After incubation for 15 min at 37°C, the reaction was stopped by adding 1.25 ml of chloroform/methanol (4:1, v/v) followed by adding 0.125 ml of water and 6 nmol of 1,2-dimyristoyl PC as internal standard. The lipids in the organic phase were extracted and analyzed by a Quattro Micro tandem quadrupole mass spectrometer (Micromass, Manchester, UK) equipped with an electrospray ion source, as described previously (46). Lipid extracts were reconstituted in 2:1 chloroform/methanol, and the samples were introduced by means of a flow injector into the electrospray ion source chamber at a flow rate of 4 l/min in a solvent system of acetonitrile/methanol/ water (2:3:1; v/v) containing 0.1% (v/v) ammonium formate (pH 6.4). The mass spectrometer was operated in the positive and negative scan modes. The flow rate of the nitrogen drying gas was 12 liters/min at 80°C. The capillary and cone voltages were set at 3.7 kV and 30 V, respectively.

Preparation of Mouse Embryonic Fibroblasts and Cell
Viability Assay-Mouse embryonic fibroblasts (MEFs) were obtained from E13.5 embryos and cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum (Biowest, Nuaille, France), 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin (Invitrogen), and maintained in 5% CO 2 . Cells were plated at 2ϫ10 4 /well in 96-well plates and then exposed to varying concentrations of tert-butylhydroperoxide (t-BuOOH, Sigma) for 6 h. Eighteen hours after treatment, cell viability was measured using 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4disulfophenyl)-2H-tetrazolium monosodium salt (WST-8), which is converted to water-soluble formazan by mitochondrial dehydrogenase (Cell Counting Kit-8, Dojindo, Kumamoto, Japan). The relative number of surviving cells was determined in quadruplicate by estimating the value of untreated cells as 100%. To determine the distribution of PAF-AH (II) between the cytosol and membranes in MEFs, MEFs were harvested after the t-BuOOH treatment (100 M) and disrupted by sonication. Cell lysates were centrifuged at 100,000 ϫ g for 1 h at 4°C to separate the cytosol (supernatant) and membrane (pellet) fractions. Both the cytosol and membrane fractions (7 g protein) were analyzed by immunoblotting with the monoclonal antibody TI10 as described above.
Measurement of 8-Isoprostaglandin F 2␣ -8-Isoprostaglandin F 2␣ (8-iso-PGF 2␣ ) was measured as previously described (49). Liver was homogenized (Polytron homogenizer, PT-3100) with saline (liver/saline ϭ 1:3, w/w), and the aliquot (300 l) was diluted with saline (1700 l). Internal standard 8-iso-PGF 2␣ -d 4 (100 ng) and 1 ml of methanol were added to this solution followed by the reduction of hydroperoxides with excessive amount of sodium borohydride at room temperature for 5 min under nitrogen. Then the reduced sample was mixed with 1 M KOH in methanol (1 ml) under nitrogen and incubated for 30 min in the dark at 40°C in a shaker. The sample was centrifuged (3000 ϫ g, for 10 min, at 4°C), and the supernatant was diluted with 4-fold volume of water (pH 3) and acidified (pH 3) using 2 N HCl. The acidified sample was centrifuged (3000 ϫ g, for 10 min, at 4°C), and the supernatant was subjected to the following solid phase extraction. C 18 cartridge was preconditioned with 2 ml of methanol and 2 ml of water (pH 3). The sample was loaded to the cartridge at a flow rate of 1 ml/min, and the cartridge was washed with 10 ml of water (pH 3) and 10 ml of acetonitrile/water (15:85, by volume). An elution was performed with 4 ml of hexane/ethyl acetate/isopropyl alcohol (30:65:5, by volume) at a flow rate of 1 ml/min. The elute was then applied at a flow rate of 1 ml/min to NH 2 cartridge preconditioned with hexane (5 ml). The cartridge was washed sequentially with 5 ml of hexane/ethyl acetate (30:70, by volume) and 5 ml of acetonitrile, and an elution was performed with 5 ml of ethyl acetate/methanol/acetic acid (10:85:5, by volume) at a flow rate of 1 ml/min. The solution was evaporated under nitrogen, and 30 l of the silylating agent, N,O-bis(trimethylsilyl)trifluoroacetamide, was added to the dried residue. The solution was vigorously mixed by a vortex mixer for 1 min and incubated for 60 min at 60°C to obtain the trimethylsilyl esters and ethers. This solution was diluted with 70 l of acetone, and then an aliquot of this sample was injected into the gas chromatograph (GC 6890 N, Agilent Technologies Co. Ltd.) equipped with a quadrupole mass spectrometer (5973 Network, Agilent Technologies Co. Ltd.). A fused-silica capillary column (HP-5MS, 5% phenyl methyl siloxane, 30 m ϫ 0.25 mm, Agilent Technologies Co. Ltd.) was used. Helium was used as the carrier gas at a flow rate of 1.2 ml/min. Temperature programming was performed from 60 to 280°C at 10°C/min. The injector temperature was set at 250°C, and temperatures of the transfer line to the mass detector and ion source were 250 and 230°C, respectively. Electron energy was set at 70 eV. The identification of 8-iso-PGF 2␣ was conducted by their retention times and mass patterns (m/z ϭ 571, 481), and ions at 481 were selected for quantification 8-iso-PGF 2␣ using the internal standard 8-iso-PGF 2␣ -d 4 (m/z ϭ 485).
Carbon Tetrachloride (CCl 4 )-induced Acute Hepatic Injury-Mice were injected intraperitoneally with 0.3 or 1 ml/kg body weight of CCl 4 dissolved in olive oil (total volume 10 ml/kg body weight). At 0, 12, 24, 36, 48, and 72 h after injection, blood was collected by retro-orbital bleeding under light ether anesthesia with heparinized capillary tubes. Plasma was obtained by cen-trifugation at 1800 ϫ g for 10 min. Plasma alanine transferase activity was evaluated using a Transaminase CII Testwako kit (Wako Pure Chemical Industries, Osaka, Japan). At each time point livers were excised and subjected to histological examination.
Statistics-Statistical analyses were performed with Student's t test setting the significance at p Ͻ 0.05.

PAF-AH (II) Is Expressed in Epithelial
Tissues-Western blot analysis showed that PAF-AH (II) was expressed essentially in all the tissues, most abundantly in the liver, kidney, intestine, and testis (Fig. 1A). Immunohistochemical studies revealed that PAF-AH (II) was predominantly expressed in epithelial cells such as hepatocytes, kidney tubules, Bowman's capsule epithelium, and intestinal column epithelium (Fig. 1B). In contrast, endothelial and interstitial cells showed low expression of PAF-AH (II). No staining of PAF-AH (II) was detected in the tissues of null mutant mice (Fig. 1B). Immunofluorescence double staining of kidney sections with antibodies to PAF-AH (II) and nephron segment markers (aquaporin-1 and aquaporin-2) revealed that PAF-AH (II) was expressed in proximal tubules, distal tubules, and thick ascending limbs of Henle but not in the collecting ducts or thin loops of Henle (supplemental figure).
Generation of Pafah2 Ϫ/Ϫ Mice-To investigate the role of PAF-AH (II) in vivo, we generated PAF-AH (II) mutant mice by targeted disruption. Mice lacking the PAF-AH (II) gene (Pafah2) were produced by homologous recombination in embryonic stem cells using the strategy outlined in Fig. 2. The targeting vector was constructed to replace exons 8 -9 of the Pafah2 gene with a neomycin-resistance gene. Targeted embryonic stem cell clones and subsequent germ line transmissions were detected by PCR and Southern blot analysis (Fig. 2B). Western blot analysis of kidney homogenates revealed that PAF-AH (II) protein was absent in homozygous mutants, and heterozygous mutants expressed about half the amount of PAF-AH (II) protein detected in wild-type mice (Fig.  2C), suggesting that the expression of PAF-AH (II) from the normal allele is not altered due to decreased amounts of total PAF-AH (II) protein. Quantitative real-time PCR analysis of kidney mRNA revealed that Pafah2 mRNA was absent in homozygous mutants (data not shown). Together, these results indicate that the introduced mutation eliminated expression of PAF-AH (II). The mutant allele was inherited in a Mendelian fashion, and the expected numbers of wild-  type, heterozygous, and homozygous offspring were born and survived to adulthood. Equal numbers of male and female pups were obtained in crosses between Pafah 2ϩ/Ϫ males and females. Mice lacking the Pafah2 gene were outwardly normal. Various organs from Pafah2 Ϫ/Ϫ mice were further examined histologically, and no abnormalities were detected by light microscopy (data not shown).

PAF-AH Activity in Pafah2
Ϫ/Ϫ Mice-PAF-AH activities were almost abolished in liver and kidney of Pafah2 Ϫ/Ϫ mice but were not reduced in brain or plasma (Fig. 3A). These results are consistent with previous results that plasma PAF-AH is responsible for most PAF-AH activity in plasma (50) and that type I PAF-AH is a major PAF-AH in brain (45) and indicate that other PAF-AHs are not up-regulated to compensate for the loss of PAF-AH (II).
We also examined the hydrolytic activities toward 1-palmitoyl 2-azelaoyl PC, one of the major oxidized phospholipids generated in vivo (51)(52)(53), in the liver and kidney of Pafah2 Ϫ/Ϫ mice. As shown in Fig. 3B, the hydrolytic activity in liver and kidney of Pafah2 Ϫ/Ϫ mice was only half that of wild-type mice, indicating that PAF-AH (II) contributes significantly to the hydrolysis of oxidized phospholipids and that another enzyme(s) is responsible for the remaining activity in these tissues.
Murine peritoneal macrophages were previously shown to metabolize PAF when cells were incubated with [acetyl-3 H]PAF, and the aqueous degradation products of PAF gradually accumulated in the culture medium in a time-dependent manner (47). Using this system we investigated the effect of deficiency of PAF-AH (II) on PAF degradation by murine peritoneal macrophages. PAF degradation by Pafah2 Ϫ/Ϫ cells was the same as wild-type cells, whereas PAF-AH activity of the homogenates of Pafah2 Ϫ/Ϫ cells was about half compared with that of wild-type cells (Fig. 4).
Sensitivity of Embryonic Fibroblasts from Pafah2 Ϫ/Ϫ Mice to Oxidative Stress-MEFs were prepared from Pafah2 Ϫ/Ϫ mice to compare their sensitivity to an oxidative stressor with that of wild-type MEFs. PAF-AH (II) was expressed in MEFs from wild-type mice (Fig. 5B) but not in MEFs from Pafah2 Ϫ/Ϫ mice  (data not shown). No obvious difference in shape or growth rate was observed between wild-type and Pafah2 Ϫ/Ϫ cells (data not shown). Pafah2 Ϫ/Ϫ MEFs were much more sensitive to the oxidative stressors t-BuOOH than wild-type (Pafah2 ϩ/ϩ ) MEFs (Fig. 5A). We previously showed that PAF-AH (II) translocates from the cytosol to membranes with oxidant treatment, whereas it translocates from membranes to the cytosol with anti-oxidant treatment (38,41). Translocation of PAF-AH (II) from the cytosol to membranes by ultraviolet radiation has also been observed in skin keratinocytes (41). Consistent with these previous studies, PAF-AH (II) translocated from the cytosol to membranes in MEFs obtained from wild-type mice upon t-BuOOH treatment (Fig. 5B).
Pafah2 Ϫ/Ϫ Mice Have Impaired Recovery from CCl 4 -induced Hepatic Injury-After treatment of wild-type control mice with CCl 4 , plasma alanine transaminase levels peaked at 48 h after treatment and then decreased (Fig. 7A), reaching normal levels by 120 h. A similar pattern was observed in Pafah2 Ϫ/Ϫ mice, except that plasma alanine transferase levels were significantly higher at the peak and in the recovery phase compared with the wild-type mice (Fig. 7A). The degree of hepatic necrosis in histological sections at 72 h after CCl 4 injection also increased in Pafah2 Ϫ/Ϫ mice (Fig. 7B). These results suggest that recovery from hepatic damage was impaired in Pafah2 Ϫ/Ϫ mice. Liver mRNA levels of Cyp2e1, a gene involved in CCl 4 bioactivation and CCl 4 -mediated liver injury (55), did not differ between wild-type and Pafah2 Ϫ/Ϫ mice (data not shown).

DISCUSSION
In this study we established PAF-AH (II)-deficient mice to analyze the physiological and pathological function of this enzyme in higher animals. Although PAF-AH activity was almost abolished especially in the liver and kidney of Pafah2 Ϫ/Ϫ mice, Pafah2 Ϫ/Ϫ mice developed normally and were phenotypically indistinguishable from wild-type mice.
PAF-AH is thought to be mainly involved in the degradation of PAF and oxidized phospholipids. Ohshima et al. (47) demonstrated that degradation of PAF by murine peritoneal mac-  rophages in culture requires at least in part PAF receptor-mediated endocytosis and the subsequent intracellular hydrolysis of the acetyl moiety. We performed the same experiments using peritoneal macrophages prepared from Pafah2 Ϫ/Ϫ mice. Although PAF-AH activity in the soluble fraction of the macrophages was about half that in wild-type mice, no change of PAF degradation was observed in Pafah2 Ϫ/Ϫ mice-derived macrophages (Fig. 4). These results suggest that PAF-AH (II) is not involved in the degradation of extracellular-derived PAF by murine macrophages. Extracellular PAF may not be accessible to PAF-AH (II) present in the cytoplasm. Alternatively, the in vitro PAF-AH assays were performed using 80 M PAF as a substrate (45), and the K m value of PAF-AH (II) is 13.5 M (24), which is extremely high compared with the PAF concentration in the culture (2 nM). Under these circumstances, the involvement of PAF-AH (II) in the extracellular-derived PAF degradation appears to be minimal.
As for the PAF-AH (II) proposed role in the degradation of oxidized phospholipids, we observed in this study that MEFs prepared from Pafah2 Ϫ/Ϫ E13.5 embryos were more susceptible to t-BuOOH than wild-type MEFs. We previously showed that overexpression of PAF-AH (II) in CHO-K1 cells suppresses t-BuOOH-induced oxidative damage (38). Travers and co-workers (41), using recombinant retroviral strategy to overexpress PAF-AH (II) in the human keratinocyte-derived cell line HaCaT, demonstrated that overexpression of PAF-AH (II) protects HaCaT cells against apoptosis induced by t-BuOOH and ultraviolet B radiation. These results suggest that the level of PAF-AH (II) is one of the determinants of the sensitivity to oxidative stress. Phospholipids oxidation products, such as 1-hexadecyl-2-azelaoyl PC, were recently shown to induce apoptosis by activating the intrinsic mitochondrial apoptotic cascade (56). Because our previous study also suggests that t-BuOOH-induced cell death occurs via apoptosis (38), it is possible that PAF-AH (II) prevents cells from oxidant-induced apoptosis by hydrolyzing such oxidatively fragmented phospholipids.
In the CCl 4 -induced acute hepatic injury model, Pafah2 Ϫ/Ϫ mice exhibited impaired recovery from tissue injury. CCl 4 is metabolized by liver microsomal cytochrome P450 -2E1 (CYP2E1) to the trichloromethyl radical (CCl 3 ⅐) (55). It incorporates O 2 to form the trichloromethylperoxyl radical (Cl 3 COO⅐) and then withdraws allylic hydrogens from polyunsaturated fatty acids to initiate lipid peroxidation (57). Antioxidants are recognized to scavenge free radicals and may, therefore, prevent propagation of the CCl 4 -induced lipid peroxidation process. Supplementation of an antioxidant such as vitamin E is known to suppress the CCl 4 -induced increase in the plasma levels of liver injury markers such as alanine transferase or asparagine aminotransferase (58 -62). Especially, the initial increase of plasma transaminase is suppressed by vitamin E treatment (58). Although the rise of plasma alanine transferase in response to CCl 4 was nearly the same in Pafah2 Ϫ/Ϫ mice and wild-type mice, plasma alanine transferase levels at the peak and in the recovery phase were much higher in Pafah2 Ϫ/Ϫ mice than in wild-type mice (Fig. 7A), indicating that PAF-AH (II) protects against CCl 4 -induced hepatic injury mainly in the later phase in contrast to vitamin E. Although the mechanism underlying these processes is not known, the accumulation of oxidized phospholipids in cells may delay the recovery from oxidative stress-induced tissue injury. We have recently reported that transgenic mice expressing PAF-AH (II) under the control of a neuron-specific promoter are highly resistant to reperfusion injury after focal cerebral ischemia, in which oxidative stress is thought to be involved (63). These results, taken together with the present results, indicate that PAF-AH (II) plays a protective role in oxidative stress-induced tissue injury in vivo.
F 2 -isoprostanes are the most reliable biomarkers of non-enzymatic lipid peroxidation and oxidative stress (64). It has been generally considered that F 2 -isoprostanes are initially formed in situ from arachidonoyl chains attached to phospholipids and then released in the free form into the circulation (65,66). Stafforini et al. (24) recently showed that PAF-AH (II) hydrolyzes esterified F 2 -isoprostanes in vitro. Because F 2 -isoprostanes are initially formed in situ from esterified arachidonic acids in phospholipids (65), we can estimate the content of esterified 8-iso-PGF 2␣ in phospholipids by subtracting the amount of free 8-iso-PGF 2␣ from the total amount 8-iso-PGF 2␣ . In this study we showed that total 8-iso-PGF 2␣ levels in CCl 4 -treated liver of Pafah2 Ϫ/Ϫ mice were significantly higher than those of wildtype mice, whereas free 8-iso-PGF 2␣ levels were not changed (Fig. 6). These results indicate that esterified 8-iso-PGF 2␣ levels were increased in Pafah2 Ϫ/Ϫ mice and also imply that accumulation of oxidized phospholipids was more pronounced in the liver of Pafah2 Ϫ/Ϫ mice after CCl 4 administration than that of wild-type mice because of impaired oxidized phospholipidshydrolyzing activity. There was no significant difference in the 8-iso-PGF 2␣ levels of liver (Fig. 6), plasma, and urine (data not shown) between untreated wild-type and Pafah2 Ϫ/Ϫ mice. As shown in Fig. 3B, a significant level of hydrolytic activity toward oxidized phospholipids still remained in the liver of Pafah2 Ϫ/Ϫ mice. These data suggest that the contribution of PAF-AH (II) to 8-iso-PGF 2␣ metabolism is not large under normal conditions or that other oxidized phospholipid-hydrolyzing enzymes compensate for the lack of PAF-AH (II).
PAF-AH (II) was predominantly expressed in epithelial cells such as kidney tubules, intestinal column epithelium, and hepatocytes, whereas endothelial and interstitial cells showed low expressions. PAF-2, a C. elegans homolog of PAF-AH (II), is also expressed at high levels in epithelial cells such as epidermal cells and intestinal cells, suggesting an evolutionarily conserved expression of PAF-AH (II) in epithelial cells from nematodes to mammals (40,41). The loss-of-function mutation in C. elegans PAF-AH (II) causes lethality due to aberrant epithelial morphogenesis during the embryogenesis (40). Despite the conservation of enzymatic properties and expression patterns in mouse and C. elegans, Pafah2 Ϫ/Ϫ mice were apparently normal. This may be because mice have certain factors that are not present in C. elegans and that compensate for the loss of PAF-AH (II) in Pafah2 Ϫ/Ϫ mice. The expression pattern of PAF-AH (II) in kidney segments (supplemental figure) is very similar to that of the components of the thioredoxin system such as thioredoxin, thioredoxin reductase, and peroxiredoxin (67). The expression pattern of peroxiredoxin 6, which can reduce phospholipid hydroperoxides, is very similar to that of PAF-AH (II) (68). It is, thus, possible that these antioxidant systems function to compensate for the loss of PAF-AH (II) in epithelial cells in mice.
Isoprostanes are also known to function in the free form as pathophysiologic mediators during oxidant injury (69). For example, 8-iso-PGF 2␣ is a potent renal vasoconstrictor, being active in the low nanomolar range (70,71). Isoprostanes are produced from both free arachidonic acids and arachidonoyl acyl chains attached to membrane phospholipids by a noncyclooxygenase mechanism involving lipid peroxidation. Because most isoprostane produced under oxidative stress is in an esterified form (Ref. 65 and in this study), it is possible that the release of isoprostanes from phospholipids is a key step for the above-mentioned biological action. This reaction may be catalyzed by PAF-AH (II). We are now testing this hypothesis.