CVR-2011-330R2
Supplementary Methods
1. Mouse studies
All experimental protocols were approved by the Hospital for Sick Children and the Toronto
Centre for Phenogenomics Animal Care Committees (AUP#0067) in accordance with the
Canadian Council on Animal Care and the Guide for the Care and Use of Laboratory Animals
published by US National Institutes of Health (NIH Publication No. 85-23, revised 1996; A504701). Alk1+/- mice were generated and genotyped as described1, and backcrossed onto the
C57BL/6 background (N4-N7; 3-18- weeks-old). In one series of experiments, 3-week-old
Alk1+/- and control littermates drank water containing 1mM Tempol (4-hydroxy-2,2,6,6tetramethypiperidine-1-oxyl, Fluka, Steinheim, Germany) for 6 weeks.
For cardiac measurements, mice were anaesthetized with 1.5% isoflurane and maintained with a
1% dose throughout the procedures. Peak right ventricular systolic pressure (RVSP) was
measured by Millar 1.4F Mikro-tip pressure transducer catheterization of the right ventricle (RV)
via the external jugular vein. In separate experiments, left ventricular systolic and diastolic
pressures were measured by left ventricle (LV) catheterization via the right carotid artery using
the 1F Millar Pressure-Volume Conductance System. Stroke volume and cardiac output were
estimated using the Millar PVAN software 3.1 and multiple-point cuvette calibration of volume
vs. conductance performed. RV was dissected from LV and septum, and RV/(LV + septum)
weight ratios calculated.
2. Vessel measurements by X-Ray Micro-CT
Mice anaesthetized with 1.5% isoflurane were intubated and breathing supported using a
pressure-controlled ventilator. Anasthesia was maintainted using 1% isofluorane. Mice were
1
CVR-2011-330R2
perfused at 20 mmHg via the RV with warm heparinized PBS followed by Microfil (Flow Tech
Inc) at 40 mmHg, using a pressure Servo System PS/200. Specimens were scanned at an
isotropic resolution of 29 µm using a Micro-CT scanner and 3D volume data reconstructed using
the Feldkamp algorithm for cone beam CT geometry as described.2 The pulmonary arterial
internal diameters of the first three-generation vessels (main pulmonary artery considered first
generation) were measured using Display and Amira software (TGS Inc, Berlin, Germany).
Images were oriented to clearly determine vessel wall boundaries.
3. Organ bath studies of isolated pulmonary arteries
Mice were anesthetized by intraperitoneal injection of ketamine (100mg/kg) and xylazine
(10mg/kg) and heart and lungs removed. Third generation lung intralobar pulmonary artery ring
segments (diameter 80-100 m; length ~2 mm) were dissected and mounted in a wire myograph
bathed in Krebs-Henseleit buffer at 37oC and bubbled with air/6% CO2 as described.2 Pulmonary
vascular muscle contraction was induced with U46619 (newborn; synthetic analog of PGH2) or
phenylephrine (adult;1-adrenergic agonist). Pulmonary vascular muscle relaxation was
induced with the endothelium-dependent agonist acetylcholine, at concentrations equivalent to
75% of the maximal agonist-induced contraction (EC75). The non-specific NOS inhibitor N (G)nitro-L-arginine methyl ester (L-NAME) was used at 100 nM concentration. Vessels were also
pre-incubated with PEG-catalase (200 units/ml) or 1mM Tempol for 20 minutes prior to addition
of acetylcholine.
4. ROS and NO determination
2
CVR-2011-330R2
Nuclear DHE staining: Freshly excised lungs were cryopreserved in OCT, snap frozen in liquid
N2 and stored at -80˚C; parallel sections (15-20 µm) were cut and slides stored at -80˚C. For
DHE staining, slides were equilibrated for 20 min at 23˚C in the dark in PBS/1% FCS, rinsed
twice in PBS and co-stained at 37˚C for 30 min with FITC-conjugated antibody to mouse CD31
(PECAM-1;1:500 dilution) and 5µM dihydroethidium (DHE, Molecular Probes). Slides were
washed twice in PBS, mounted and immediately imaged in parallel using the GFP and CY3
channels for CD31-FITC and DHE respectively, on a TE2000 inverted fluorescent microscope.
H2-DCFDA assay: Lungs were homogenized in phosphate-buffered Krebs containing 1mM
NaN3 and centrifuged at 13000 g for 15 min. ROS levels in the supernatants were measured at
37˚C using 10 µmol/L 5-(and 6-)-carboxy-2-,7-dichloro dihydrofluorescein diacetate (carboxyH2DCFDA, Molecular Probes, Invitrogen, Burlington, ON). Fluorescence was quantified with a
SpectraMax Gemini EM spectrofluorometer using 488 nm excitation and 525 nm emission
wavelengths. Levels were normalized for protein content with the Bradford assay.
H2O2 microsensor: Lung homogenate supernatants and plasma were tested using a calibrated
H2O2 specific sensor (Word Precision Instruments, Saratosa, FL) attached to an Apollo 4000
Free Radical Analyzer. Results are expressed as µmoles H2O2/g protein for lung tissues and nM
H2O2 concentration in plasma.
Amplex Red assay: Amplex Red (Molecular Probes) was used to measure H2O2 levels in lung
homogenates according to manufacturer’s instructions. Freshly dissected lungs were
homogenized in 25 mM HEPES buffer, pH 7.4, plus 1 mM EDTA and protease inhibitors. After
centrifugation at 6000 g for 5 min at 4°C, the supernatants were incubated in a microtiter plate at
37°C for 1 h with Amplex red, horseradish peroxidase and L-Arginine (NOS substrate, 1 mM,
Sigma-Aldrich, Oakville, ON). The effects of 1mM L-NAME and 0.05 mM antimycin (a
3
CVR-2011-330R2
respiratory chain reaction inhibitor, Sigma-Aldrich) on the reaction were tested. Fluorescence
was quantified using 544 nm excitation and 590 nm emission and levels normalized for protein
content.
NO measurements: Freshly excised lungs were cut in small fragments and incubated in KrebsHenseleit buffer for 2 h at 37°C, with and without L-NAME. NO was quantified using a NO
microsensor ISO-NOP700 attached to an Apollo 4000 Free Radical Analyzer. The microsensor
was calibrated using S-Nitroso-N-acetylpenicillamine (SNAP) in the presence of copper sulfate.
5. eNOS phosphorylation and Western blot analysis
For eNOS phosphorylation assays, fresh lung tissues were excised, cut into 5-mm segments,
equilibrated for 90 minutes in serum-free MCDB 131 media supplemented to 10 mM MgCl2 and
with 10 M ATP. Tissue samples were treated with or without 1 mM L-NAME for 15 min and
then incubated with vehicle or 1 μM ionomycin for 10 min. Samples were washed in PBS,
homogenized as described below and pre-cleared with protein A/G mixture.
For all Western blots, lung tissues were homogenized in 10 mM Tris-HCl pH 7.4 lysis buffer
containing 1% Triton X-100 and protease/phosphatase inhibitors (Roche), and centrifuged at
13,000 g for 30 min. Equivalent amounts of lysate proteins in Laemmli buffer were fractionated
on 4-12% gradient SDS/PAGE and immunoblotted. The following primary antibodies were used:
Nox4, rabbit IgG, 1:200 (Lab Vision Co Fremont, CA); eNOS, 1:3000; gp91phox/Nox2, 1:1000;
and COX-2, 1:500 (mouse IgG1, BD Biosciences, Mississauga, Ontario); SOD1, 1:4000;
Catalase, 1:2000; SOD3/EC-SOD, 1:3000; and Alk1, 1:1000 (goat IgG, R&D, Minneapolis,
MN); SOD2, rabbit IgG, 1:3000 (Lifespan Biosciences Seattle; -actin, 1:40000 (SigmaAldrich); endoglin, rat IgG2a clone MJ7/18, 1:1000 (SouthernBiotech, Birmingham, AL);
4
CVR-2011-330R2
phospho-Ser-1177 eNOS and phospho-Thr-495 eNOS, (1:1000) (rabbit IgG; Cell Signaling
Technology, Danvers, MA).
Appropriate IgGs conjugated with horseradish peroxidase
(1:20000 dilution) were used as secondary antibodies. The enhanced chemiluminescence (ECL,
Perkin Elmer, Shelton, Connecticut, USA) reagent was used for detection. Band intensities were
quantified and expressed relative to -actin.
References
1.
Seki T, Yun J, Oh SP. Arterial endothelium-specific activin receptor-like kinase 1
expression suggests its role in arterialization and vascular remodeling. Circ Res 2003;93:682689.
2.
Belik J, Jerkic M, McIntyre BA, Pan J, Leen J, Yu LX et al. Age-dependent endothelial
nitric oxide synthase uncoupling in pulmonary arteries of endoglin heterozygous mice. Am J
Physiol Lung Cell Mol Physiol 2009;297:L1170-1178.
3.
Toporsian M, Jerkic M, Zhou YQ, Kabir MG, Yu LX, McIntyre BA et al. Spontaneous
adult-onset pulmonary arterial hypertension attributable to increased endothelial oxidative stress
in a murine model of hereditary hemorrhagic telangiectasia. Arterioscler Thromb Vasc Biol
2010;30:509-517.
5