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SUPPLEMENTAL MATERIALS AND METHODS
Animals. All animal protocols were reviewed and approved by the Institutional Animal Care and
Use Committee of Kanazawa Medical University. Specific-pathogen-free (SPF) male
Sprague-Dawley (SD) rats were purchased from Japan Charles River Inc. (Yokohama, Japan) and
were housed in the SPF rat room of the Kanazawa Medical University Laboratory Animal Center.
Reagents. Unless otherwise specified, all common chemicals were purchased from Sigma (St.
Louis, MO) or Wako Chemicals (Osaka, Japan). Oleic acid (OA) was obtained from Sigma (O1008).
Antibodies were purchased as follows: a mouse monoclonal anti-5×His tag antibody from Novagen
(Darmstadt, Germany), a mouse anti-rabbit GAPDH antibody from Chemicon (Temecula, CA), a
rabbit anti-human prosurfactant protein C (proSP-C) from RDI (Flanders, NJ), and a goat anti-rabbit
IgG (Alexa 488) from Abcam (Cambridge, UK), a horseradish peroxidase-conjugated goat
anti-mouse IgG antibody from Bio-Rad (Hercules, CA). Commercial kits were purchased from as the
following: ApopTag-Red Apoptosis in situ Detection Kit (Fluorescence S7156) from Millipore
(Temecula, CA), Secretory phospholipase A2 Assay kit from Abcam, Rat CXCL1/CINC-1
Immunoassay kit from R&D Systems (Shanghai, China), rat myeloperoxidase ELISA kit (HK105)
from Hycult Biotech (Plymouth Meeting, PA), and OxiSelect In Vitro ROS/RNS Assay kit from Cell
Biolabs (San Diego, CA). A chemiluminescent detection kit, a stripping buffer, and a
micro-bicinchoninic acid (BCA) protein assay kit were obtained from Pierce (Rockford, IL). A rat
inflammatory ELISA kit was purchased from Qiagen (Valencia, CA). A TLC apparatus was obtained
from Advantec (Tokyo, Japan), and Silica gel G plates were obtained from Analtec (UniplateTM,
Newark, DE).
Cloning of mouse LPCAT1-cDNA. Total lung RNA was extracted from a male C57BL/6J
mouse lung that had been treated with RNAlaterTM (Ambion, Austin, TX) using the RNeasy reagent
(Qiagen), followed by treatment with RNase-free DNase I. Total single-stranded lung cDNAs were
synthesized with the SuperScript III assay kit (Invitrogen, Carlsbad, CA) using oligo(dT)20 , followed
by treatment with DNase-free RNase. NCBI BLAST searches were performed to design two primer
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sets for the cloning of mouse LPCAT1-cDNA (GenBank accession number AB244717.2) in two
fragments. One of these sets consisted of a forward primer containing an EcoR1 restriction site and
ATG initiation codon with a six-histidine tag:
5’-CCGGAATTCACCATGCATCATCATCATCATCATATGAGGCTGCGGGGCCGCGGGC-3’,
and a reverse primer: 5’-ACTGTGCCCGTCGCTTGA-3’. The other set consisted of the forward
primer: 5’-CGGCTCCTGTTCGCTGCTT-3’, and a reverse primer that contained a stop codon and a
Hind3 restriction site: 5’-CCCAAGCTTCTAGTCCGCTTTCTTACAAGAATTC-3’. PCR was
performed with KOD DNA polymerase (KOD FX) (TOYOBO, Osaka, Japan) using the following
program: 5 cycles of 96 °C for 15 s, 40 °C for 30 s, and 68 °C for 45 sec, followed by 30 cycles of
96 °C for 15 s, 60 °C for 30 s, and 68 °C for 45 sec. The two PCR products were cloned separately
by TA cloning (10×A-attachment mix) (TOYOBO), and digested with two separate restriction
enzyme pairs. One of the products was digested with EcoR1 and BstE2, yielding a 0.46-kb fragment.
The other was digested with BstE2 and Hind3, yielding a 1.2-kb fragment. These two fragments
were inserted together into the shuttle plasmid pDC316 (Microbix, Toronto, Canada) using T4 DNA
ligase. DNA sequencing was performed on the recombinant plasmid pDC316-LPCAT1 with the
BigDye terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA) and confirmed
the presence of precisely the same nucleotide sequence as the mouse LPCAT1.
Construction of recombinant adenovirus. The adenovector construction kit (AdMaxTM) and
low passage HEK293 cells were purchased from Microbix. HEK293 cells are human embryonic
kidney cells transformed by sheared adenovirus type 5 DNA, and these cells contain/express the
early region of adenovirus type 5. They complement the growth of E1-defective adenovirus mutants
and vectors. The reconstructed adenoviruses lack the E1 region which is necessary for replication;
thus, they can efficiently infect alveolar epithelial cells but not replicate in these cells except for
E1-bearing cells such as 293 cells (1). The shuttle plasmid pDC315 was digested with BamH1 and
Sal1. The lacZ-cDNA was excised from the pRSET/lacZ plasmid (Invitrogen) with BamH1 and
Hind3, then inserted into the pBlueBacHis2A plasmid (Invitrogen), digested again with BamH1 and
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Sal1, and reinserted into the pDC315. All plasmids including the adenovirus genomic plasmid
(pBHGGloxE1,3Cre) were purified with CsCl-density gradient ultracentrifugation. 293 cells were
co-transfected with one of the recombinant shuttle plasmids (lacZ or LPCAT1) and the adenovirus
genomic plasmid (pBHGGloxE1,3Cre) in the presence of CaCl2. Recombinant virus plaques
appeared in 2 − 3 wk. The expression of lacZ and LPCAT1 was confirmed through a -galactosidase
enzyme assay in live 293 cells and Western blotting conducted in 293 cell lysate with a mouse
anti-5×His tag monoclonal antibody, respectively.
LPCAT1 enzyme assay. Microsomal cell fractions were prepared from ~ 3.2×106 of 293 cells
or ~10×106 of freshly isolated rat ATII cells as previously reported (2). The LPCAT1 enzyme assays
were performed in 0.1 ml of a liposome mixture (100 mM Tris-HCl, pH 7.4, 1 mM EDTA)
containing 1 mg/ml DPPC, 0.25 M [1-14C]palmitoyl-CoA (3.70-5.55 MBq/mmol) (ARC0516,
1.48-2.22GBq/mmol), 0 to 100 M 1-palmitoyl-lysoPC, and 1 g of microsomal protein prepared
independently at different experiments. The mixture was incubated at 30°C for 5 min, and then the
reaction was stopped by adding 0.3 ml of CHCl3:MeOH (1:2). Total lipids were extracted by the
Bligh-Dyer method (3), and subjected to one-dimensional TLC with a developing solvent
(CHCl3:MeOH:H2O=70:30:5, v/v). The bands corresponding to LPC and PC were scraped off and
were counted with a liquid scintillation counter.
Culture of ATII cells. ATII cells were isolated from male SD rats at the age of 8 − 10 wk as
previously reported (4), and 1.5 ×106 cells were then seeded at in 1 ml of 5% rat serum (RS)
(Pel-Freez Biologicals, Rogers, AR)/DMEM per 30-mm Millicell-CM culture insert (Millipore) with
2 ml of the same medium added outside the insert. The inner inside bottom surface of the insert had
previously been coated with 0.4 ml of a 4:1 (vol/vol) mixture of rat tail collagen and
Engelbreth-Holm-Swarm (EHS) tumor matrix (MatrigelTM) (Collaborative Biomedical Products,
Bedford, MA). After overnight culture at 37 oC in a 10% CO2 incubator, cells were infected with the
adenovector at multiplicity of infection (MOI) = 10 in DMEM for 1 hr. The cells were replenished
with 0.4 ml of complete medium (DMEM/5% rat serum/1% charcoal stripped FBS/10 ng/ml
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KGF/10-8M dexamethasone) within the insert and 2 ml outside the insert. When indicated, the cells
were radiolabeled with 0.5 Ci of [3H]choline chloride/ml of the complete medium. The six-well
plates were then placed on a rocking platform inside a humidified incubator gassed with 10% CO2.
The plates were rocked approximately six times per min. After 2 days of culture on the rocking
platform, the cells were used for experiments. The cells were treated with 0, 0.1, 0.5, or 1 mM of
hydrogen peroxide in warmed DMEM and cultured for exactly 1 hr, then immediately placed on ice
to stop the reaction, and washed twice with cold PBS. When indicated, 0.15% Trypan blue in PBS
was added, followed by incubation on ice for exactly 5 min. The cells were then rinsed with cold
PBS twice and fixed with cold 4% paraformaldehyde/PBS. Under a light microscope at a magnitude
of 400×, 200 cells were counted, and the percentage of Trypan blue dye-stained cells was expressed
as the %dead cells. In another experiment, ATII cells radiolabeled with [3H]choline were washed
vigorously with DMEM twice, and total cellular lipids were then extracted by the Bligh-Dyer
method (3). The extracted total cellular lipids were subjected to one dimensional TLC, and
[3H]-labeled PC and [3H]-labeled LPC were quantified on one-dimensional TLC.
Western blotting. A fixed amount of proteins from 293 cells lysed with a lysis buffer [150 mM
NaCl, 50 mM Hepes (pH 7.4), 10% glycerol, 1.5 mM MgCl2, 1 mM EGTA, pH 7.4, 1%
Triton-X100] containing antiproteinases (1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinin) was
subjected to 4−12% Bis-Tris SDS-PAGE under reducing conditions and then transferred to a
nitrocellulose membrane. The membrane was immunoblotted with a mouse anti-5×His tag
monoclonal antibody (1:1,000) or a mouse anti-GAPDH antibody (1:10,000) for 1 h at 37°C,
followed by incubation with a horseradish peroxidase-conjugated goat anti-mouse IgG antibody
(1:5,000) for 1 h at 37°C. A chemiluminescent detection assay was performed, and the obtained
bands were developed using an autofluorography film.
Phospholipid assay. At 4 hr after OA injection, the lavaged left lungs were excised and
subjected to phospholipid analysis. Total lipids were extracted from the BALF and lavaged left lung
tissues according to the Bligh and Dyer method (3). In all cases, 20% of each sample was used for
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phospholipid phosphorus content determination following the method described by Bartlett (5). The
remaining 80% of each sample was subjected to two-dimensional TLC (6). After development, the
phospholipid species were visualized using iodine vapor. The spots were then scraped off, and
phospholipid quantities were determined.
Adenovector delivery to rat lungs. Male SD rats at 8- to 10-wk of age were used in these
experiments. In the viral treatment experiment, rats were anesthetized through an intraperitoneal
administration of pentobarbital sodium (~ 15 mg) and intubated using a 16G venous catheter.
Through this catheter, an extra-thin soft polypropylene catheter connected to a 1-ml syringe
containing adenovector was inserted into the lung, and a purified high-titer adenovirus [109
plaque-forming units (pfu)] in 0.5 ml of solvent (0.45% NaCl/2.5% glycerol)] or the same volume of
solvent alone was slowly administered. The efficiency of the transfection of lung cells by a single
endobronchial administration of Ad-lacZ was evaluated by ex vivo endobronchial staining of the
lungs in lacZ assays. Seven days post-administration of the adenovector, the lungs were excised,
fixed, and stained for 4 hr via intratracheal administration of 0.5 mg/ml X-gal (1). The lung sections
were embedded in paraffin, sectioned, and counter-stained with nuclear fast red (Kernechtrot).
Induction of acute lung injury (ALI) by oleic acid. At 1wk post-adenovector
pre-administration, the rats were anesthetized as described as above. The trachea was exposed and a
16G venous cannula was intubated. A 125 l/kg body wt dose of pure OA was mixed with 0.1%
BSA/saline to obtain a total volume of 0.2 ml, followed by vigorous vortexing. Through a femoral
vein, either the OA mixture or the same volume of a 0.1%BSA/saline solution was slowly injected
over 1 min, followed by 0.2 ml of 0.1%BSA/saline to flush the syringe and needle. The rats were
kept under anesthesia for 4 hr with spontaneous breathing, and then exsanguinated by severing the
abdominal aorta. The left lung was lavaged with a 2.5 ml of HBS (0.9% saline/10 mM Hepes, pH
7.4) five times. This procedure yielded approximately 12 ml of bronchoalveolar lavage fluids
(BALFs). Following lavage, the left lung was excised, and the lower half of the lung was subjected
to phospholipid analysis. The right middle and lower lobe (containing an accessory lobe) were
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ligated and excised en-bloc for measurement of the wet-to-dry lung weight. The excised lungs were
gently blotted and weighed and then dried at 85°C in an oven for 3 days until exhibiting a constant
weight. The right upper lobe was fixed via an intratracheal administration of a 10% neutral formalin
solution at a constant pressure of + 25 cmH2O.
In other experiments, the right intermediate bronchus was ligated, cut off, weighed, snap-frozen
with liquid nitrogen, and stored at – 80 oC until use. The right upper lobe and left lungs were lavaged
with a 3.0 ml of HBS five times. This procedure yielded approximately 14 ml of BALFs. Following
lavage, the right upper lobes were cut off, weighed, and snap-frozen. The left lung was excised, and
fixed with 10% neutral formalin, and used for immunofluorescence staining of the lung tissues.
Cytokines, enzymes, and reactive oxygen/nitrogen species assay. The BALFs were
centrifuged at 180×g for 10 min, and the supernatant and cell pellet were separated. ELISA was
performed in these BALF supernatants using an ELISA kit for rat inflammatory cytokines including
IL-1, IL-1, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, interferon , TNF, GM-CSF, and RANTES
according to the manufacturer’s protocol. The rat CXCL1/CINC-1 concentration in the BALFs was
measured independently using another ELISA kit.
The frozen right lungs were rapidly thawed and, while cooled in ice water, these lungs were
homogenized with a Polytron-type homogenizer in four volumes (weight) of PBS. The homogenates
were centrifuged at 12,000 ×g for 10 min, and the supernatants were recovered and separated into
several aliquots. The myeloperoxidase (MPO) amount, secretory phospholipase A2 (sPLA2) activity
(thio-PC release, mol/min), and reactive oxygen/nitrogen species (ROS/RNS) (RFU: relative
fluorescence unit) were measured using commercial assay kits according to the manufacturer’s
protocols.
Immunofluorescence staining of the lung tissues. Formalin-fixed and paraffin-embedded left
lungs were cut at a thickness of 5 m, and the sections were used for double-fluorescence staining of
apoptosis and pro-surfactant protein C (proSP-C). Apoptosis was detected by the TUNEL [terminal
deoxynucleotidyl transferase(TdT)-mediated dUTP nick end-labeling] method according to the
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manufacturer’s protocol with a minor modification. Antigen retrieval was performed using 20 g/ml
of proteinase K treatment for 15 min. After incubation with TdT for 1 hr, the tissue section was
incubated with a rabbit anti-human proSP-C antibody (7) at 1:1000 dilution for 1 hr, followed by
incubation with a rhodamine-conjugated sheep anti-digoxigenin antibody and an
Alexa488-conjugated goat ant-rabbit IgG antibody at 1:400 dilution for 30 min. The specimens were
observed with a fluorescence microscope (Keyence BZ-9000, Osaka, Japan) and ×40 magnifying
object lens. More than 10 alveolar fields in the lung tissue section prepared from one rat were
randomly selected. Number of green-stained ATII cells and ATII cells with red-stained apoptotic cell
nuclei were counted, and apoptosis ratio of ATII cells in the lung field was calculated.
Lung histology. The right upper lobes of the lungs, inflated with 10% neutral formalin at a
constant pressure of +25 cmH2O, were paraffin-embedded, sectioned at a thickness of 5 m to
include the largest cross-sectional area of the lobe, and stained with hematoxylin and eosin (H&E).
Observation fields were randomly selected under a light microscope at a magnification of 400×,
along with the pleura. Twenty to twenty-five peripheral alveolar fields per lung were independently
evaluated by two pulmonary pathologists according to the acute lung injury scoring system (8), with
no information about the experimental groups on the lung slides.
Lung mechanics. Rats were anesthetized and OA was intravenously injected as described above.
Anesthesia was maintained for 4 hr with spontaneous breathing. The trachea was exposed and
cannulated with a 16G catheter attached to a three-way stopcock. The rats were mechanically
ventilated with 100% O2 (~ 2.0 ml tidal volume and ~ 60 cpm) (Harvard model 683, South Natick,
MA) for 5 min, followed by a 5 min closure of the trachea to completely degas the lungs (9). The rats
were exsanguinated and the anterior chest and diaphragm were extensively dissected away. The
lungs were connected to a syringe pump (Minato MCIP-IIIS, Tokyo, Japan) and a semi-conductor
pressure transducer (Kulite XCW-190-5D, Leonia, NJ) through the three-way stopcock; the lungs
were then inflated continuously with air at 3.0 ml/min to a maximal pressure of 30 cmH2O and
allowed to equilibrate for 30 sec. The lungs were then deflated at the same speed down to −5 cmH2O.
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This inflation-deflation cycle was repeated 3 times, and the fourth cycle was used for analysis. Care
was taken not to cause air leak from the lungs. When an air leak was found, the lungs were discarded.
Pressure-volume curve was plotted and used to assess lung compliance, which was defined as the
slope of the linear region of the deflation limb where pressure ranged from 0 to 5 cmH2O. The lung
compliance was normalized by body weight.
Statistics. For parametric samples, all data were expressed as mean ± S.D. The equality of
variances was verified with F-test between two populations, or Bartlett test for more than three
populations. For comparisons of the mean values between two experimental groups, Student’s t-test
was employed (Figs. S1B). Single-factor ANOVA was employed for comparisons among three
experimental groups (Fig. 1), or two-factor factorial ANOVA for four experimental groups (Figs. 2, 3,
4, 5B), and followed by Student-Newman-Keuls post hoc test for multiple comparisons. In some
cases, data were log transformed because of their sample distributions (Fig. 4: CXCL1/CINC-1, IL-6,
TNF, and MPO). Non-parametric acute lung injury (ALI) scores were expressed as median ±
25th/75th percentile. Data were evaluated with Kruskal-Wallis test to verify the difference in the mean
values among four experimental groups, followed by Steel-Dwass test for non-parametric all-pairs
multiple comparisons (Fig. 5D). In all cases, a p value less than 0.05 was considered statistically
significant. Significance between the Ad-lacZ group and the Ad-LPCAT1+OA group or the
Ad-LPCAT1 group and the Ad-LacZ+OA group was not shown because of their less importance.
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REFERENCES
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adenovector-mediated gene transfer of keratinocyte growth factor on the proliferation of alveolar
type II cells in vitro and in vivo. Am J Respir Cell Mol Biol 2000; 23: 626-635.
2. Nakanishi H, Shindou H, Hishikawa D, Harayama T, Ogasawara R, Suwabe A, Taguchi R,
Shimizu T. Cloning and characterization of mouse lung-type acyl-CoA:lysophosphatidylcholine
acyltransferase 1 (LPCAT1) Expression in alveolar type II cells and possible involvement in
surfactant production. J Biol Chem 2006; 281: 20140-20147.
3. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem
Physiol 1959; 37: 911-917.
4. Dobbs LG, Mason RJ. Pulmonary alveolar type II cells isolated from rats. Release of
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5. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 1959; 234: 466-468.
6. Poorthuis BJ, Yazaki PJ, Hostetler KY. An improved two dimensional thin-layer chromatography
system for the separation of phosphatidylglycerol and its derivatives. J Lipid Res 1976; 17:
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7. Vorbroker DK, Voorhout WF, Weaver TE, Whitsett JA.
Posttranslational processing of
surfactant protein C in rat type II cells. Am J Physiol 1995;269:L727-33.
8. Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS, Kuebler WM.
Acute Lung Injury in Animals Study Group. An official American Thoracic Society workshop
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SUPPLEMENTAL FIGURE LEGEND
Figure S1. Ad-LPCAT1-infected 293 cells express recombinant LPCAT1 protein. (A) Western
blot showing 6×histidine-tagged LPCAT1, with a molecular weight of ~ 60 kDa. 293 cells
were infected with Ad-lacZ or Ad-LPCAT1 at an MOI of 10. Two days later, the cells were
lysed and a 50g of post-nuclear cell lysate proteins was subjected to Western blot analysis
using a mouse anti-5×His tag monoclonal antibody (upper) or a mouse anti-GAPDH
antibody (lower) as the primary antibody. (B) The recombinant 6×histidine-tagged
LPCAT1 protein expressed in 293 cells reveals high activity of the LPC acyltransferase
enzyme. The acyltransferase activity of LPCAT1 was measured in the presence of 1 g of
the microsomal fraction obtained from Ad-LPCAT1-infected 293 cells (closed circles),
Ad-lacZ-infected 293 cells (open triangles), intact rat ATII cells (closed square), or intact
293 cells (cross). The measured values were normalized to those obtained in ATII cells. n
= 3 independent experiments, *P<0.05, **P<0.01, ***P<0.001 vs. Ad-lacZ.
Figure S2. Efficiency of the transfection of lung cells by a single endobronchial administration
of Ad-lacZ evaluated by ex vivo endobronchial staining in lacZ assays. Seven days
post-administration of Ad-lacZ or 0.45%/2.5% glycerol, the lungs were excised, fixed, and
stained for 4 hr with an intratracheal administration of 0.5 mg/ml X-gal (1). (A) The left
lung after endobronchial staining by a lacZ assay for 4 h. (B) The stained lung was
embedded in paraffin, sectioned, and counter-stained with nuclear fast red (Kernechtrot)
stain. Magnification, 200×. Bar = 40 m.