CIRCULATIONAHA/2007/743997/R2
Online data supplements
Materials.
LY374388 ([3-Aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol-4-yloxy]-acetic acid methyl ester) is a prodrug of LY329722 (sodium [3-aminooxalyl-1-benzyl-2-ethyl-6-methyl-1H-indol-4-yloxy]-acetic acid), an inhibitor of sPLA2 activity. Both LY374388 and LY329722 were from Shionogi Research Laboratories (Osaka, Japan). The peritoneal administration of LY374388 at 100 mg/kg yielded 5~10 mol/L plasma concentration of LY329722 over 12 h after administration. The IC50 of LY329722 for mouse sPLA2-X and human sPLA2-X, sPLA2-V, sPLA2-IIA, and sPLA2-IB activities were 3.1, 22, 63, 14, and 313 nmol/L, respectively. The purified recombinant mouse sPLA2-X protein and the rabbit anti-human sPLA2-X polyclonal antibodies were prepared as in our previous report.1 Anti-mouse neutrophils polyclonal antibody was from Cedarlane (Ontario, Canada)and anti--tubulin polyclonal antibody was from Santa Cruz Biotechnology (Santa Cruz, USA). Cell culture reagents were from Sigma (Tokyo, Japan) and Invitrogen Corp. (Carlsbad, USA). Other chemicals were purchased from Sigma unless otherwise indicated.
Mouse.
The experimental protocol was approved by the University of YamanashiAnimalCare and Use Committee, and procedures were carried out inaccordancewith the National Institutes of Health Guide for the Care and Use ofLaboratory Animals (1996). All sPLA2-Xmiceanalyzed were male and fromF13~ F15(10-14 weeks-old, 20 to 25 g), and the littermate sPLA2-X male mice were used as controls. sPLA2-receptor mice were generated as in our previous report2 and sPLA2-receptor male mice from F13 ~ F15 and the littermate sPLA2-receptor male mice were used.
Construction of targeting vector and generation of sPLA2-X-deficient mice.
The mouse sPLA2-X gene was cloned from a 129 SVJ genomic library (Stratagene) using oligo DNA, 5’-tacacctgtcactctcctatgtgtg-3’ of mouse sPLA2-X as a probe. The 2.8-kb Xba I-Acc II and 3.0-kb Pst I-Pst I genomic fragments derived from the isolated clone were used for the construction of the targeting vector, along with a neomycin-resistance gene driven by the phosphoglycerete kinase-1 (Pgk-1) promoter (Pgk-neor) and a diphtheria toxin A-fragment gene driven by the MC1 promoter as positive and negative selection markers, respectively (see the supplemental FigureI).3 Using this construct, homologous recombination results in the replacement of the Acc II-Pst I genomic fragment that includes the translation starting codon in the Pgk-neor cassette, resulting in abolition of sPLA2-X expression.
The ES cell line used in this study was E14 derived from 129/Ola.4 The targeting experiment and generation of mutant mice were performed essentially as described previously.5 In brief, the E14 cells (1.7×107 cells) were electroporated with a Bio-Rad Gene Pulser (0.8 kV, 3 microfarads) using 30 g of NotI-linearized targeting vector. The electroporated cells were selected in medium containing G418 (125 g/mL). The cells of surviving colonies were screened for homologous recombination by Southern blot analysis. The mutant cells were microinjected into 3.5-day-old C57BL/6J blastocysts, and the embryos were transferred into the uteri of pseudopregnant ICR mice. The germline chimera was backcrossed for 10 generations with C57BL/6 mice to obtain sPLA2-Xmice with a C57BL/6 background. The resulting sPLA2-XF13 ~ F15 mice were then intercrossed to generate the homozygous sPLA2-Xmice. The genotypes of mice were determined by Southern blot analysis of DNA prepared from tails.Genomic DNAs were digested with PvuII overnight and electrophoresed through 0.8% agarose gels. The DNAs were transferred to GeneScreen Plus membranes (NEN Life Science Products) and probed with a 0.36-kb fragment amplified by PCR.
Bone marrow transplant experiments.
sPLA2-X mice and sPLA2-X mice (10-14 weeks of age) were sacrificed by cervical dislocation.The marrow from the tibias and femurs was harvested under sterileconditions. Bones were flushed with DMEM with 10% FBS. The marrow was passed through a 25-gauge needle in order to obtain single-cell suspensionsof bone marrow cells. Cells were washed and followed by hypotoniclysis of erythrocytes, yielding approximately 20 x 106 nucleated bone marrow cells per mouse. The sPLA2-X+/+mice (7-8 weeks of age) were lethally irradiated with 8 Gy. Immediately after irradiation, 5 x 106bone marrow cells from sPLA2-X+/+mice or sPLA2-Xmice were injected through the tail vein. At 6 weeks after the transplantation, the efficiency of reconstitution was evaluated. More than a totalof 95% of peripheral leukocytes had been reconstitutedas determined by real-time quantitative PCR for peripheral white blood cells genomic DNA. The resulting chimericmice were subjected to the myocardial ischemia-reperfusion experiments in vivo, as described below.
Measurements of mRNA and protein expression levels.
Total RNA was extracted from tissues and cells with a Qiagen RNeasy kit and DNase I (QIAGEN, Tokyo, Japan). The mRNA expression levels were quantified by a real-time two-step reverse transcriptase PCR assay using SYBR Green I chemistry and with the use of a 7500 Real-Time PCR System (Applied Biosystems, Foster City, USA).6 The PCR primers are listed in the supplemental Table I. The GAPDH housekeeping gene was used for normalization of gene expression.
For immunoblot analysis, 15 g protein of the extracts of neutrophils, myocardium, lung, and spleen, or 15 Lof serum isolated from mouse after ischemia-reperfusion injury or the supernatant from the isolated neutrophils and plateletsafter activation were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane.6 The intensity of the -tubulin band was used as a loading control between the tissue samples.
ELISA for sPLA2-X.
The 96-well plates were coated with rabbit anti-human sPLA2-X polyclonal antibody (IgG, 200 ng), blocked with 1.0% skimmed milk, and incubated with various concentrations of samples in 0.4% skimmed milk for 2 h at room temperature. After washing 3 times with PBS/0.05% Tween 20, the plates were incubated with rabbit anti-sPLA2-X polyclonal antibody (IgG) conjugated with biotin in 0.4% skimmed milk (1 g/mL) for 1 h.1 The plates were washed and incubated with streptoavidin-conjugated horseradish peroxidase for 45 min. The signals were detected with tetramethylbenzidine. The specific signals were calculated as OD450 values after subtracting the background obtained with the plates coated with nonimmunized rabbit IgG. In this assay, the purified recombinant mouse sPLA2-X was used as a standard protein that was detectable in the range 1 pg/mL to 20 ng/mL.
Immunohistochemistry and immunofluorescence of mice heart.
Hearts were harvested after ischemia-reperfusion and were fixed in 10%formalin solution, embedded in paraffin and then sliced into sections of 5 mthickness.6 These sections were stained by the ABC technique (Vectastain ABCkit, Vector Laboratories, Burlingame, USA) and a sequential double-labeling method6with anti-human sPLA2-X polyclonal antibody 1and anti-mouse neutrophil polyclonalantibody as primary antibodies andwith biotinylatedanti-rabbit IgGas secondary antibodies(Vectastain ABCkit, Vector Laboratories). For immunofluorescence, a part of myocardial sections were incubated with the primary antibodies followed by the secondary antibodies. The primary antibodies includedanti-human sPLA2-X antibody, anti-mouse neutrophil polyclonalantibody, and anti- -actinin antibody (Sigma). The secondary antibodies for the immunofluorescence study included Alexa Fluor 488, Alexa Fluor 546, and Alexa Fluor 647 (Invitrogen). The slides were examined using a confocal microscope(Olympus FV-1000, Tokyo, Japan) equipped with a x40/1.0-numerical aperture oil-immersionobjective. In some sections, the neutralized sPLA2-X polyclonal antibody with a 10-fold excessof mouse sPLA2-X proteinwas used as primary antibody to examine the specificity of the immunoreactivity with the anti-human sPLA2-X polyclonal antibody.
Myocardial ischemia and reperfusion in vivo.
The sPLA2-X mice, the sPLA2-X mice, or the chimeric mice were subjected to 1 hof myocardial ischemia and 24 h of reperfusion.6 Briefly, mice were anesthetized with pentobarbitalsodium (50 mg/kg body wt). Mice were intubated and ventilated on a respirator (model SN-480-7, Shinano, Tokyo, Japan) under electrocardiographic (ECG) monitoring (surface ECG, lead II). Ischemia was achieved by ligating the left anteriordescending coronary artery (LAD) using an 8-0 silk suture witha section of PE-10 tubing placed over the LAD, 1 mm from thetip of the normally positioned left atrium. After occlusionfor 1 h, reperfusion was initiated by releasing the ligatureand removing the PE-10 tubing. Successful coronary occlusion and reperfusion were demonstrated by visual changes in the ischemic region and significant ECG changes.The chest wall was closed, theanimal extubated. During the procedure, body temperature was maintained by use ofa 37°C warming plate. Hearts were harvested at 24 h of reperfusion.The loosened suture was left in place and then retied for thepurpose of evaluating the ischemic area.
When the effect of LY374388, a prodrug of an inhibitor of sPLA2, on the myocardial infarct size was examined in the sPLA2-X mice, LY374388 or vehicle was administered once intraperitoneally at a dose of 100 mg/kg 1 h before the ischemia or at the beginning of the reperfusion.
Assessment of area at risk (AAR) and infarct size.
After 1 hof myocardial ischemia and 24 h of reperfusion, the LAD was reoccluded at the same position and 0.5ml of 1% Evans blue dye was administered through a 26 G needle inserted into the LV.After removing, the left ventricle (LV) was cut transversely into 5 sections and each section was then weighed and incubated in 1% triphenyltetrazolium chloride (TTC) for 10min at 37°C. At the completion of TTC staining, viable myocardium was stained brick red and the infarct remained a pale white. After photographing each slice, computerized planimetry (NIH Image J analysis software) was used to analyze all sections of the slices, including AAR and infarcted areas. The sizes of the AAR and infarcts in proportion to the total size of the slices were calculated and multiplied by the weight of each slice to determine the AAR and infarct weight per slice. Infarct size was expressed as a percentage of LV mass and a percentage of AAR.
Langendorff experiment.
Mice were heparinized (5000 U/kg body wt) and anesthetized (pentobarbital sodium50 mg/kg body wt, i.p.). Hearts were excised rapidly and mounted on a Langendorff perfusion system at 37°C and perfused at 80 mm Hg with Krebs-Henseleit buffer gassed with 95% O2/5% CO2 (pH 7.4). A water filled polyethyleneballoon connected to a pressure transducer was inserted into theLV. The balloon was inflated to adjust the LV end-diastolic pressure (LVEDP) to~10mm Hg. The hearts were paced at 420 beats/minby means of platinum wires placed on the right atrium. After a stabilization period, the hearts were subjected to 20 min of no-flow global normothermic ischemia followed by 60 min of reperfusion. The LVEDP, left ventricular developed pressure (LVDP) (peak systolic pressure minus LVEDP), maximum rate of contraction (+dP/dt), and maximum rate of relaxation (−dP/dt) were monitored during the experiments using a Power Lab data acquisition system (AD Instruments, Boulder, CO). Coronary flow rate was measured by timed collection of the coronary effluent. LDH released into the coronary effluent during the reperfusion period was measured with a commercially available assay kit (Sigma) and expressed as units per 60 minutes of the reperfusion period per gram of wet heart weight (U/60 min/g).Finally, hearts were then perfused with 3 ml of 1% TTC at 37°C for 10 min at pH 7.4.Then, LV was cut transversely into 5 sections, weighed, and photographed. The infarct size was also assessed by the computer planimetry.
In vivo transthoracicechocardiography.
M-mode echocardiography was performed at baseline and after 1 hof ischemia followed by 24 h of reperfusionunder ketamine/xylazineanesthesia using a 15-MHz phased-array probe connected to a Sonos 5500 echocardiograph (Philips Medical Imaging, Andover, Massachusetts). In brief, an M-mode cursor waspositioned in theparasternal short-axis view perpendicular to the interventricular septum and posteriorwall of the LV at the level of the papillary muscles, and M-modeimages were obtained for measurement ofLVend-diastolic and end-systolic dimension (LVDd and LVDs). The percentage of fractional shortening (%FS)was calculated from the equation %FS = [(LVDd – LVDs)/LVDd]x 100.All data were averaged from 5 cardiaccycles/experiment.The individuals who performed the echocardiography and theresulting calculations were blinded to the genotypes of the mice.
Measurement of myeloperoxidase (MPO)activity and neutrophil counts in infarcted hearts.
The MPO assay procedures are similar to methods described in our previous report.7 Hearts were exercised after 1 h of ischemia followed by 3 h of reperfusion and divided into ischemic and non-ischemicparts. The ischemic tissues were weighted and homogenized and sonicatedin 0.5%hexadecyltrimethyl-ammonium bromide in 50mmol/Lpotassium phosphate buffer, pH 6.0.The mixture was centrifugedat 12,500 rpmfor 30min at 4°C. The supernatants were then collectedand reacted with 0.167mg/mL of -dianisidine dihydrochlorideand 0.0005% H2O2 in 50mmol/L phosphate buffer at pH 6.0.The change in absorbance was measured spectrophotometrically at460nm. One unit of MPO activity is defined as the quantityof enzyme hydrolyzing 1mmol peroxide/min at 25°C.
Midventricular tissue slices were preparedfrom the ischemic parts subjected to 1 h of ischemia followed by 3 h of reperfusion. Histological staining (hematoxylin and eosin) was performedon multiple sections of the ischemic tissue to determinethe extent of neutrophil infiltration in ischemic/reperfused myocardium. For each of the hearts examined,neutrophils were identified bynuclear morphology in 6 fields of 5 separate sections by a blinded observerand are presented as neutrophils/mm2.
Preparations and counting of neutrophils and other blood cells in peripheral blood and bone marrow.
Mice were injected intraperitoneally with 2 mL of 2% casein in sterile saline. Mice were sacrificed by cervical dislocation 4 h after the intraperitoneal injection. The saline was injected into the peritoneal cavity, then the peritoneal fluid was collected. The cells were precipitated by centrifugation (650 g, 5 min). Neutrophils were further purified from the peritoneal cells by Ficoll-Paque gradients followed by hypotoniclysis of erythrocytes.7Thenumber of neutrophils recovered from one mouse was 1~2 x 107 cells and the purity was > 98% by Türk stain (0.01% of methilrosaniline chlorideand 1.0% acetic acid), neutrophil alkaline phosphatase and neutrophilesterase stain. After 1 h of suspension in serum-free Medium 199 at 4°C, the isolated neutrophils were used in the experimental protocols described below. Citrated blood was obtained by intracardiac puncture under the anesthesia. Platelet rich plasma was obtained by centrifugation at 240 g for 12 min.Neutrophils were also harvestedfrom the bone marrow oftibias and femurs after killing by cervical dislocation and were isolated by the Ficoll-Paque gradients as described above.A part of blood samples obtained by cardiac puncture were transferredto microtainer tubes containing the anticoagulant EDTA. Samples were analyzed for red blood cell, whiteblood cell, and platelet counts byBürker-Türk cell counter, and peripheralblood smears were stained with May-Giemsa, and neutrophils number was counted by morphology(100 cells counted/slide). Bone marrow differentials were similarly performed in the smears of cytospin preparations of flushed marrow cells.
Respiratory burst function in neutrophils.
Production of active oxygen metabolites during neutrophil activation was measured by the method of luminol-dependent chemiluminescenceusing the Luminescencer-PSN AB-2200 (ATTO, Japan), as described in our previous report.7 The isolatedneutrophils (1×106 cells/mL) were suspended in a cuvette with serum free Medium 199withoutphenol red at 37°C. The light emission was recorded after addition of luminol (0.1 mmol/L), followed bythe addition ofPBS as a vehicle or one of the activators including C5a (2 g/mL), fMLP (10 mol/L), A23187 (2 mol/L), arachidonic acid (AA, 1 mol/L), or phorbol-12-myristate-13-acetate (PMA, 10 mol/L). In some experiments, neutrophils were preincubated for 3 min with mouse sPLA2-X protein (0.1 ~ 1 ng/mL), LY329722 (10 nmol/L), or PBS or DMSOas a vehicle. Integrated counts were determined as the sum of chemiluminescence counts integrated for 20 min after activation.
Neutrophil migration assay.
Migration assayswere performed using a modified 48-well Boyden chamber. Incubationlasted 30 min in a 5% CO2/95% atmosphere at 37°C. After incubation,cells on the upper surface were removed by scraping,and cells adherent to the lower side of the filter were fixed inmethanol and stainedfor counting. PBS as a vehicle or one of the chemotactic compounds including C5a (2 g/mL), fMLP (10 mol/L), A23187 (2 mol/L), arachidonic acid (1 mol/L), or PMA (10 mol/L) was placed in thelower wells of the Boyden chamber. In some experiments, mouse sPLA2-X protein (0.1 ~ 1 ng/mL) or PBS as a vehicle was also placed in the lower wells together with the chemotactic compound. LY329722 (10 nmol/L), a sPLA2 inhibitor, was added to both upper and lower chambersin one set of experiments to examine its inhibitory effect on neutrophil migration.
Neutrophil elastase release.
Isolated neutrophils were suspended in serum-free Medium 199without phenolred and activated withserum-opsonized zymosan(OZ, 1 mg/mL) and then pelletedby centrifugation (15,000 rpm for 20min). The hydrolytic activityof neutrophil elastase in the harvested supernatants was determinedusing synthetic substrate Suc-Ala-Pro-Ala-MCA.7
Arachidonic acid release from isolated neutrophils.
The release ofarachidonic acid in response to C5a (2 μg/mL) and fMLP (10μmol/L)was measured using neutrophils prelabeled with 0.5 μCi/mL [3H] arachidonic acid.1 Theradioactivity in the culture medium was expressed as a percentage of the totalradioactivity incorporated into the cells.In some experiments, neutrophils were preincubated for 3 min with LY329722 (50 nmol/L) or DMSO asa vehicle.
Preparation and culture of mouse cardiomyocytes.
Primary cultures of adult mouse cardiomyocytes were prepared by collagenase digestion from the ventricles of 10- to 14-week-old mice as described previously.8 Briefly, adult hearts from sPLA2-Xmice were rapidly excised and placed on a cannula for perfusion through the aorta. Perfusion was performed for 5 minwith PBS including 10 mmol/L 2,3-Butanedione monoxime (BDM), followed for 10 min with perfusion buffer containing Liberase blendzyme 1(0.25 mg/mL), trypsin (0.14 mg/mL), and CaCl2 (12.5 mol/L). After mechanicaldissection of the left ventricle cells, the cardiomyocytes were resuspended with myocyte plating MEM medium containing 5% FBS, BDM (10 mmol/L), and L-glutamine (2 mmol/L) and plated on laminin (BD Biosciences, FranklinLakes, USA)-coated culture dishes. After incubation at 37 °C, 2% CO2 for 1 h, the medium was exchanged to myocyte culture MEM medium (0.1 mg/mL BSA, 10 mmol/L BDM, 5 g/mL insulin, 5 g/mL transferrin, and 5 ng/mL selenium). Each experiment using cultured cardiomyocytes was performed 2 h after the incubation.
Effects of recombinant mouse sPLA2-X protein on cultured mouse myocardial cells.
The cultured mouse myocardial cells from sPLA2-Xmice were treated with PBS as a vehicle or mouse sPLA2-X protein at the indicated concentrations for 24 h.Thereafter, cell viability was assessed by measurement of two enzymatic activities: mitochondrial dehydrogenase activity by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method, and lactate dehydrogenase (LDH) activity in the supernatant medium by a commercially available LDH kit. For the MTT assay, 200 L of MTT solution was added to 1 mL of culture medium after the treatment. After the incubation at 37 °C for 4 hrs, cells were solubilized with solubilization solution. The absorbance at 570 nm of each aliquot was determined using a microplate reader. Thereduction in MTT activity in the treated cells was presented as percentage of non-treated control cells. LDH activity was measured in the collected culture medium and the harvested cells with the lysis buffer according to the manufacturer’s protocol. The percentage of LDH released into the medium was calculated by dividing the amount of activity in the medium by the total activity (medium LDH and cellular LDH).Also, the cytotoxic effects of sPLA2-X protein were compared between sPLA2-receptor (R) and sPLA2-R myocardial cells (sPLA2-X genotype).2In some experiments, extracellular release of arachidonic acid in response to mouse sPLA2-X was measured using cultured mouse myocardial cells prelabeled with 0.5 µCi/mL[3H] arachidonic acid.1 The radioactivity in the culture medium was expressed as apercentage of the total radioactivity incorporated into thecells. In some experiments, the culturedcardiomyocytes were incubated withthe indicated concentrations of mouse sPLA2-X proteinor PBS as a vehicle in the presence of LY329722 (10 nmol/L) or DMSO asa vehiclein a hypoxia chamber (Ikemoto scientific technology, Tokyo, Japan) filled with 1% O2/2% CO2/ 97% N2 at 37°C for 12 h, followed by reoxygenation by placing the cells in normoxic condition (98% atmosphere/2% CO2) for 12 h. Thereafter, cell viability was assessed with MTT assay.