Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
Supplement
Supplementary methods and results
Human studies
Exclusion criteria for the enrolment in this trial were acute or chronic infection, critical
disorders such as acute coronary syndrome, uncontrolled hypertension, heart failure,
pregnancy, age below 18 years, known allergy to nicotine, and participation in another
clinical trial within the last 3 months. 30 persons with normal MPO expression and activity
and matched for age, gender, smoking status, cardiovascular risk factors, and medication
served as a control group.
Vascular function tests
Vascular function studies were performed by two blinded examiners in the morning after an
overnight fast and following 10 minutes of resting. Methods of assessing flow-mediated
dilation followed the principles set by the international brachial artery reactivity task force.1
Endothelial function was assessed in the subjects' right arm in a quiet, temperature-controlled
room (22°C) by high resolution ultrasound (Sonoline G50, 12 MHz linear array transducer,
Siemens). Longitudinal scans of the brachial artery were obtained approximately 5 cm
proximal of the antecubital fossa. The transmit focus zone was set at the depth of the anterior
wall. Anatomical landmarks and snapshot images were used to assess flow-mediated dilation
in the exact same vessel section at each time point. 2D cine-sequences of the brachial artery
over 4 s at baseline and 1 minute after induction of reactive hyperemia by 5-minute cuff
occlusion of the forearm (50 mmHg over the systolic blood pressure) were recorded. Mean
flow velocity was assessed by pulsed wave Doppler with correction of insonation angle at
baseline and peak hyperemic flow to calculate the flow ratio. 2D sequences were analyzed
using an edge detection software (Brachial Analyzer, Medical Imaging Applications LLC).
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Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
Image acquisition and analysis were performed separately. After 10 minutes of rest,
endothelial function was determined following sublingual application of 25 g of
nitroglycerin, according to the same recording protocol. Inter- and intraobserver variabilities
for brachial diameter measurements in our laboratory are 0.12 ± 0.10 and 0.05 ± 0.17 mm,
respectively.
Genetic analysis of MPO deficiency
DNA for genetic analysis extracted from peripheral blood leukocytes with a commercial kit
(Qiagen, Hilden, Germany) was available for 10 patients. All exons and flanking intronic
sequences of the MPO gene on 17q23 were amplified by polymerase chain reactions with
Taq-polymerase (PeqLab, Erlangen, Germany) under standard conditions. After purification,
PCR-products were directly sequenced using fluorescence based didesoxy-chain-termination
technique (Big Dye Termination kit, ABI, Germany). The primers used for generation of the
PCR-products were used for the sequencing reaction. The chromatograms were visually
inspected for ambiguities or heterozygosity and subsequently blasted against the reference
genomic assembly of chromosome 17. The reported variations were concordantly detected on
both strands.
In 4 of 10 subjects mutations were detected that accounted for MPO deficiency. Two subjects
exhibited the c.2031-2C-mutation in a heterozygous state, one was heterozygous for the
mutation c.995T (p.332Val) and one homozygous for the c.752C mutation (p.251Thr).
Animal Experiments
Sixteen domestic pigs of either sex were used for the experiments. After intramuscular
premedication
with
azaperon
(4
mg/kgBW),
midazolam
(0.3
mg/kgBW),
ketaminhydrochlorid (5 mg/kgBW), and atropinsulfat (0.15 mg/kgBW), intravenous
anesthesia was induced by pentobarbital 8mgkgBW and maintained by continuous infusion
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Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
of fentanyl (0.01 mg/kgBW/h) and midazolam (0.1 mg/kg BW/h). Pigs were endotracheally
intubated and pressure controlled ventilated at 15 cm H2O, PEEP of 7 cmH2O, and 16 breaths
per minute, using 30% oxygen. Given the documented interference of heparin with vessel
wall immobilized MPO2, bivalirudin, a direct thrombin inhibitor known to allow for binding
of MPO towards the vessel wall was administered (0,75 mg/kgBW bolus, 1,75 mg/kgBW/h
until study end-point).3
The heart was exposed through median sternotomy. For continuous flow measurements, the
proximal segment of the LAD and IMA were dissected free from surrounding tissue and a
2mm transit-time flow probe (CardioMed Flowmeter, Medi-Stim AS, Oslo, Norway) was
placed around each vessel. In order to avoid changes of hemodynamic parameters due to
changes in heart rate, an epicardial pacemaker was installed, which kept the heart rate at 100
beats per minute.
A 5F catheter was inserted into the carotid artery for continuous hemodynamic monitoring
and regular arterial blood gas analyses.
Vessel preparation and organ chamber studies
IMA segments were cut into pieces measuring 4 mm each and placed into organ chambers
filled with 25 ml of phosphate buffer and indomethacin (10 µmol/l) and fumigated with a
carbogen gas mixture (95% O2, 5% CO2 ) at a constant temperature of 37°C. Tension on the
vessels was continuously increased within the first 60 minutes up to 5 grams in order to
optimize contractions to potassium chloride (KCl 80 mmol/l). After washing and repetition
of the procedure phenylephrine (Phe) was titrated until approximately 50-70% of the
maximum contraction under KCl was achieved. Endothelial-dependent relaxation was
determined in response to increasing doses of acetylcholin (Ach: 10–9-10-6 mol/l) as
previously.4
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Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
Myocardial Blood Flow Measurements Using Fluorescent Microspheres
Approximately 4×106 fluorescent microspheres (FM), 15 µm in diameter (Molecular Probes,
Eugene, Oregon), were injected into the left atrium during withdrawal of a reference blood
sample via the abdominal aorta as previously described.5 Excitation and emission
wavelengths for each of the labeled FM did not interfere with the excitation and emission
wavelength of indocyanine green (blue 356/424 nm, blue-green 427/468 nm, yellow-green
495/505 nm, orange 534/554 nm, red 570/598 nm, crimson 612/638 nm, and scarlet 651/680
nm).
After the animal was sacrificed at the end of the experiment, hearts were excised and fixed in
10% formaldehyde for 6-8 days. The anterior wall of the left ventricle was sliced into 20
wedge-shaped transmural tissue pieces, corresponding to a reticule projected onto the FCI
computer images. Tissue samples and arterial blood reference were processed for
determination of MBF by spectrofluorometry according to the standard method described by
Glenny et al..6
Myocardial Perfusion Measurement Using Fluorescent Cardiac Imaging
Myocardial perfusion was assessed using a fluorescent cardiac imaging system (FCI) system
as described previously (LLS GmbH, Ulm, Germany).5 The fluorescent dye indocyanine
green (ICG) is intravenously applied (0.01 mg/kgBW) and rapidly binds to plasma proteins
which guarantees the intravasal remain. A short half time of 2.4 minutes due to hepatic
clearance enables repeating injections in short time intervals. The heart is illuminated with
near-infrared light at a wavelength of 785 nm with a total output of 80 mW in a field of view
of 10 cm in diameter operating at a distance of 20 cm over the heart. The excited dye shows a
fluorescence with an emission maximum at 830nm, which is detected by an infrared sensitive
CCD camera system (dynamic range 54dB) equipped with a band pass filter for selective
transmission of light at the emission maximum of ICG. Each FCI sequence was recorded online (25 frames/s) for 60 s with real-time digitizing using a frame grabber card providing a
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Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
resolution of 8 bits. Data were analyzed off-line after recording the images using a
customized software package with temporal resolution of 40 ms and spatial resolution of
about 0.2 mm at a penetration depth of 4 mm (LLS GmbH, Ulm, Germany).
To measure myocardial perfusion by FCI the slope of fluorescent intensity (SFI) derived from
the time dependent fluorescence signal was calculated, as previously.5
To obtain a quantitative measure for the fluorescence intensity both the mean value and the
standard deviation of the measured pixel intensities in the region of interest (ROI) on the
myocardial wall for each image in a sequence of 60 s after the injection of the ICG were
determined. The size of the ROI was 30x30 pixels, which corresponds to an area of about 6x6
mm on the myocardial surface depending on the distance from the camera.
SFI data were correlated to FM data in the corresponding area. Therefore the reticulated FCI
computer images were then matched with the corresponding myocardial slices of the anterior
wall of the left ventricle.
Endothelial function studies in murine aortas
To reinforce the tenet that the observed effects on endothelial function in humans were not
mainly mediated by leukocyte/MPO-independent effect direct nicotine-based effects murine
aortic segments were incubated with nicotine (10 mM), nicotine-activated leukocytes (1 hour
pre-incubation of wildtype-derived leukocytes with nicotine 10 mM) and MPO (137 pmol/l),
respectively. As a substrate of MPO, H2O2 (10 mmol) was added to all conditions.
Endothelium-dependent and -independent vasorelaxation was determined as previously.7
As shown below, incubation with nicotine did not result in a significant deterioration of
endothelial function as compared to control. In contrast, incubation with nicotine-activated
leukocytes induced a significant reduction of endothelial function and even more striking,
MPO decreased Ach-induced relaxation to the highest extent. There was no difference
between the groups regarding endothelium-independent relaxation (p=0.377).
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Rudolph et al. Myeloperoxidase deficiency preserves vasomotor function in humans
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