Online appendix
Methods:
Carotid plaque characterization
The carotid endarterectomy specimens were divided in segments of 5 mm thickness along the longitudinal axis
of the vessel. The segment with greatest plaque burden was embedded in paraffin for further histological
characterization. The directly adjacent segment was used for protein extraction by dissolving in 40 mM TrisHCl (pH=7.5) at 4°C as previously described.24
Histology of human atherosclerotic specimens
For NGAL staining, the following procedure was used: paraffin sections (5-μm thickness) were deparaffinized,
blocked for 30 minutes in 1.5% hydrogen peroxide in methanol, and boiled for 12 minutes in 10 mM sodium
citrate (pH 6.0). After pre-incubation with 10% normal rabbit serum in 1% PBSA for 1 hour, incubation with
primary antibody was performed overnight (monoclonal rat-anti-NGAL, 10ug/mL; R&D Systems). Biotinylated
rabbit-anti-rat IgG (0.4ug/mL; DAKO) was used as the secondary antibody, followed by horseradish peroxidase
conjugated streptavidin, AEC substrate and counterstained with hematoxylin. Specificity of the staining was
ascertained by absence of staining in isotype controls and increasing intensity of the staining with increasing
concentrations of the primary antibody. Co-localization of NGAL with macrophages, smooth muscle cells and
endothelial cells was assessed by double stainings. NGAL staining was performed under the same conditions as
for the single NGAL staining, except Fast Blue substrate was used, followed by CD68 (macrophage) staining
(KP-1, 3.3 μg/mL; DAKO), or Alpha-actin (1A4, 1:1500; Sigma, St. Louis, MO) or CD34 (Qbend10, 0.5
μg/mL; Beckman Coulter, Fullerton, CA) with AEC substrate. Neutrophil infiltration was assessed using
myeloperoxidase staining (polyclonal rabbit anti-human, 1:10000; DAKO).
Paraffin sections of the plaques were stained and analyzed semi-quantitatively for macrophages (CD68), smooth
muscle cells (Alpha-Actin), collagen (Picrosirius Red), calcifications (Hematoxylin and Eosin (H&E)) and
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overall phenotype, as described previously.24 Interleukin-6 (IL-6) and IL-8 were measured in plaque protein
extracts with a multiplex suspension array system according to the manufacturer’s protocol (Bio-Rad
Laboratories, California, USA). MMP-2, -8 and -9 activities were measured by Biotrak RPN2631, RPN2635
and RPN2634, respectively (Amersham Biosciences, Buckinghamshire, UK). The measurements for MMPs and
interleukins were standardized for total protein concentration of the protein extracts (DC protein assay, BioRad).
Histology of murine atherosclerotic specimens
From 4mm caudal to 5mm cranial of the right renal artery branch, 5µm (cryo) or 3µm (paraffin) sections were
made at every 0.5mm.
Oil Red O (lipid) stainings were performed on cryo-sections.
Paraffin sections (For iron, macrophage-, 24p3-, micelle and alpha-SMA staining):
Murine aorta was perfusion-fixated with formaldehyde 4%/PBS (1:10) and embedded in paraffin. Prussian Blue
staining was used for detection of iron particles on deparaffinized 3µm thick sections. For the 24p3 and MAC3
(macrophages) the sections were boiled for 20 minutes in citrate-buffer (pH=6), cooled (au bain Marie) and
washed in PBS/Tween. For alpha-SMA the sections were boiled for 20 minutes in EDTA (pH=9.0). After
incubation for 60 minutes with the primary antibody, either 24p3 (1:100, Santa Cruz, sc-50351), or MAC3
(1:200, eBioscience, 11-5989), or rabbit polyclonal to alpha-SMA (1:800, Abcam (Cambridge, UK)), the
sections were washed with PBS/Tween. The MAC3 stained sections were incubated for 60 minutes with a
secondary rabbit anti-rat antibody (DAKO, p0450) in 10% normal mouse serum and washed in PBS/Tween. All
sections were incubated for 30 minutes with PowerVision (biotin-free polyclonal-AP-anti rabbit, Immunologic,
DPVR-110AP), and subsequently washed in PBS and TBS. Then, sections were incubated for 30 minutes in
alkaline-phosphatase substrate, washed with aqua-dest and counterstained with hematoxylin. Finally, sections
were washed with flowing tap water and air-dried.
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In case of the micelle-staining, sections were deparaffinized and washed with aqua-dest, incubated for 60
minutes with rabbit anti-rat (1:250, DAKO, p0450) in 10% normal mouse serum and washed with PBS/Tween.
After incubation with Powervision (biotin-free polyclonal-AP-anti rabbit, Immunologic, DPVR-110AP) for 30
minutes, sections were washed with PBS and TBS. Sections were incubated for 30 minutes with alkalinephosphatase substrate, washed with aqua-dest and counterstained with hematoxylin. Finally, sections were
washed with flowing tap water and air-dried. All stainings were performed at room temperature.
Detection of NGAL/MMP-9 complex in carotid plaques
Gelatin zymography was performed as described before.38 An equal amount of total protein extract was loaded
for each sample38. The combined gelatinolytic activity of NGAL/MMP-9 (125 kD) and NGAL-dimer/MMP-9
(150 kD) was measured as the degradation of gelatin at 125 kD and 150 kD and is further referred to as
NGAL/MMP-9 gelatinolytic activity.14 Gelatinolytic activity of free MMP-9 (92kD) was measured and is
further referred to as MMP-9 gelatinolytic activity. Western blotting for NGAL was additionally performed
using monoclonal rat-anti-human NGAL antibody (5 μg/mL; R&D Systems) followed by biotinylated rabbitanti-rat IgG (0.675 μg/mL; DAKO, Glostrup, Denmark) and horseradish peroxidase conjugated streptavidin.
Levels of the NGAL/MMP-9 complex were determined by ELISA (R&D Systems, Minneapolis, MN). An equal
amount of protein extract was loaded for all samples. However, because the sensitivity of the ELISA was
relatively limited (lower detection limit 312 ng/mL for NGAL/MMP-9 ELISA compared to 10 ng/mL for
NGAL ELISA), only a limited subset of samples with high protein concentrations could be used for the analysis
due to loading limitations.
Baseline and follow-up MRI procedure
Scout images:
Fast Spin Echo (FSE) technique was used for axial scout images. Subsequently, coronal and saggital scout
images of the suprarenal abdominal aorta using Fast Low Angle Shot (FLASH) were acquired. Twenty
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contiguous axial slices, perpendicular to coronal and saggital scout images, were planned from 4 mm below the
right renal artery branch up to 5 mm above this branch.
Optimization of trigger delay:
Signal acquisition was planned at the moment of maximal flow through the abdominal aorta. Hereto axial T1
weighted FSE (cardiac and respiratory gated) at the level of 1 mm above the right renal artery was repeated with
varying trigger delay. Images were checked for laminar flow artifacts (bright vessel rims as a result of slow
flow) and the diameter of the aortic lumen. Trigger delay corresponding with the image, which lacked flow
artifacts and had the largest aortic diameter was chosen (optimized trigger delay) for other scans as described
below.
IR-FSE images:
IR-FSE axial images from 4 mm below the right renal artery branch up to 5 mm above were acquired. The
acquisition scheme was started with an inversion pulse. The chosen trigger delay (TD) for IR-FSE was chosen
in a way, that the equation “(inversion time + TD)/(cardiac cycle duration) = integer + optimized TD” was
satisfied.
Reference images for recognition of anatomic landmarks: lymph nodes and small peri-aortic blood vessels:
Diffusion weighted imaging sequence was used with diffusion gradient (b=400 s/mm2) and with the same
acquisition modules but zero diffusion (b=0 s/mm2). These images were used for discrimination of peri-aortic
lymph nodes (having low diffusion) usually found at 5 levels around the right renal artery branch (-1 up to +3
mm). A steady-state free precession technique was used for acquisition of non-triggered bright-blood images for
depiction of small peri-aortic blood vessels.
MRI parameters:
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IR-FSE images:
TR/TE=3500/14 ms, inversion time=1300 ms, rare factor=10, excitation/refocusing pulse gauss 1 ms, flip
angle=90˚/180˚, Spectral width=75757 Hz, FOV=30x30 mm2, matrix=200x200 resulting in a pixel
dimension=150x150 µm2, 20 axial slices, thickness=0.5 mm, chemical shift selective fat suppression, 2 inflow
saturation slices (1 cranial (saturation of arterial inflow) and 1 caudal (saturation of venous inflow)), thickness
10 mm, NA=10, zero fill acceleration factor 1.7, total acquisition time 7m20s.
Diffusion weighted images:
TR/TE=2000/13.2 ms, hermite pulse 1.0 ms, excitation/refocusing flip angle=90˚/180˚, b value (longitudinal/zaxis) 0 or 400 s/mm2, diffusion gradient duration 1.40 ms, separation 5.0 ms, Spectral width=50000 Hz,
FOV=30x30 mm2, matrix=256x256 resulting in a pixel dimension=117x117 µm2, 1 axial slice, thickness=0.5
mm (at five levels around the right renal artery: -1 up to +3 mm), chemical shift selective fat suppression,
NA=1, zero fill acceleration factor 1.9, total acquisition time 4m20s. Lymph nodes were discerned by
calculation of the apparent diffusion coefficient: ADC=ln(S1-S2)/ΔB. S1 and S2 are the mean signal intensities
on the image with b=0 and the image with b=400 s/mm2, respectively, while Δb = 400-0=400 s/mm2. If ADC
was less than 1.8, the structure was identified as a lymph node.
Bright-blood images:
TR/TE=150/1.75 ms, gauss pulse 500 µs, flip angle=90˚, Spectral width=166667 Hz, FOV=30x30 mm2,
Matrix =256x256 resulting in a pixel dimension=117x117 µm2, 20 axial slices, thickness=0.5 mm, NA=5, zero
fill acceleration factor 1.34, total acquisition time 2m24s.
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Supplement Figure Legends
Supplement Figure 1: Association between NGAL and MMP activity
A: MMP-9 activity (Biotrak) is strongly related to gelatinolytic activity of the NGAL/MMP-9 complex
(r=0.315; p=0.005) in carotid plaques. B: Representative zymogram (ZY) and NGAL Western Blot (WB),
demonstrating that the gelatinolytic bands at 125 and 150 kD contain NGAL. C: representative zymogram
detecting NGAL/MMP-9 complex at 125 kD and NGAL-dimer/MMP-9 complex at 150 kD.
Supplement Figure 2: NGAL preserves MMP-9 activity
Quantification of in vitro experiments by gelatin zymography. A: In vitro reconstruction of NGAL/MMP-9
complexes by combining recombinant NGAL and MMP-9. B: MMP-9 activity diminishes after 30 minutes
incubation at 37ºC (left lane: no incubation, right lane: 30 minutes incubation). C: Increasing addition of NGAL
prevents the autodegradation of MMP-9, (MMP-9:NGAL molar ratio 1:5 – 1:125). D: Quantification of C.
Supplement Figure 3: Histology of brachiocephalic artery atherosclerosis in apoE-/- mouse
A. EVG staining, B. NGAL staining, C. smooth muscle cell (alpha-SMA) staining.
*: buried fibrous cap suggested by an intra-plaque layer of smooth muscle cells; L: lumen.
Supplement Figure 4: Histology of murine aortic atherosclerosis in apoE-/-/eNOS-/- mouse
A. NGAL staining, B. smooth muscle cell (alpha-SMA) staining, smooth muscle cell layers suggesting buried
fibrous cap (arrow) with enlarged view of indicated region, C. iron staining (Prussian Blue). Dashed region with
positive iron staining is zoomed in D. Arrowheads: intra-plaque hemorrhage represented by blue iron staining.
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