ONLINE APPENDIX
Signaling and functional defects of high-density lipoproteins (HDL) in coronary artery
disease (CAD) are caused by low sphingosine-1-phosphate (S1P) content and can be
efficiently corrected by HDL loading with S1P
Katherine Sattler, MD*, Markus Gräler, PhD†, Petra Keul, PhD*, Sarah Weske, MSc*, Christina-Maria
Reimann, MSc†, Helena Jindrová, MSc*, Petra Kleinbongard, PhD*, Roger Sabbadini, PhD‡, Martina
Bröcker-Preuss, MD§, Erbel Raimund, MD║ (FACC), Gerd Heusch, MD* (FACC), Bodo Levkau, MD*
Supplemental Methods
Generation of plasma
Peripheral venous EDTA blood was drawn from healthy human subjects (n=70) and patients with
stable CAD (n=64) recruited consecutively to expand the groups on which our previous findings on
CAD-HDLs-S1P were based (1). Venous EDTA mouse blood was obtained by cardiac puncture.
Immediately after blood drawing, the tubes were placed on ice. Plasma was generated by
centrifugation, immediately recovered and frozen at -80°C. HDL-C in murine plasma was measured
with a commercial kit (Fluitest HDL-D, Analyticon Biotechnologies AG, Germany) and by enzymatic
methods in human plasma (Advia Chemistry Systems DHDL, CV 2.36%; Bayer Health Care,
Germany). The study was approved by the ethics committee of the University Hospital Essen and
complies with the Declaration of Helsinki. Written informed consent to participate in the study was
obtained from each participant. Demographic and clinical data are provided in Supplemental Table 1.
Protein concentration of HDL was determined by Bradford assay and cholesterol with a commercial
kit (Fluitest Chol, Analyticon Biotechnologies AG, Germany).
Isolation of high-density lipoproteins
High-density lipoproteins (HDLs), HDL2 and HDL3 were isolated from pooled plasma (mice) or
individual samples (humans) by sequential density gradient ultracentrifugation according to their
density (ρ>1.069<1.21g/mL, HDL2: ρ >1.063g/mL<1.125g/mL, HDL3: ρ >1.125g/mL<1.21g/mL (2))
1
and as already published for these two groups (1). Murine HDLs were isolated from pooled plasma
samples of several animals as indicated; human HDLs were isolated from individual plasma samples.
Protein concentration of isolated HDL was determined by Bradford assay (BioRad) and total
cholesterol concentration with a commercially available kit (Fluitest Chol, Analyticon Biotechnologies
AG, Germany), respectively.
Oxidation of HDL
Isolated HDLs were oxidized with copper sulfate (CuSO4) exactly as described (3). Briefly, HDLs
(protein concentration 1mg/mL) were incubated with a freshly prepared CuSO4 solution added to a
final concentration of 50μM at 37°C for 250 minutes (4). EDTA was added at a final concentration of
5-fold that of copper (250μM) to stop oxidation. Oxidized HDLs were dialyzed for 24 hours against
EDTA-saline (0.5mM EDTA, 150mM NaCl, pH 7.4) in the dark at 4°C with four changes of the
dialysis buffer. Optical density of the samples was measured spectrophotometrically at 245nm before
and after oxidation and after dialysis to monitor successful oxidation (4). Protein concentrations of
oxidized HDL samples were re-determined after dialysis by the Bradford assay. Oxidized HDLs were
immediately used for in vitro assays.
Measurement of S1P
S1P was measured in plasma or lipoproteins by investigators blinded for the experiments as described
(1). In brief, lipids were extracted from the samples by successive addition of 1mL of methanol, 200µl
of 6mol/L HCl and twice 2mL of chloroform. Chloroform phases were retrieved by centrifugation, and
chloroform was removed by vacuum-drying in a speed-vac. Subsequently, samples were dissolved in
100μl of methanol/ chloroform (80:20 v/v). S1P was measured by LCMS. Distribution of S1P in
plasma and in HDL was calculated as described (1) .
In vitro assays for S1P-loading of isolated HDL
Peripheral venous blood of healthy volunteers was collected in vacuum tubes filled with 16IE
heparin/mL (Sarstedt). Erythrocytes were isolated by washing the blood sample consecutively with
2
ice-cold phosphate buffered saline (PBS) and erythrocyte buffer (150mmol/L NaCl, 5mmol/L KCl,
21mmol/L Tris-HCl, 0.1% glucose, pH 7.4), and removing in each centrifugation step the white blood
cell layer. The cells were diluted to a hematocrit of 0.5 and incubated with sphingosine (final
concentration 10μmol/L) for one hour at 37°C. Erythrocytes efficiently generate S1P from sphingosine
and do not degrade but release it in the presence of an acceptor (5). After washing the suspension with
ice-cold erythrocyte buffer and dilution to a hematocrit of 0.5, HDL or human plasma were added at
the indicated concentrations and durations at 37°C under slight shaking. The reaction was terminated
by placing it at 4°C, and the supernatants were recovered by centrifugation. In a second approach
(direct loading), HDLs were loaded with S1P by adding 0.1 mg of HDL (at a concentration of
1mg/mL HDL protein) to 6 nmol of S1P (for all in vitro studies) or 3 pmol S1P (for all vasodilation
studies) after evaporation of its methanol solvent. For this, S1P dissolved in methanol (1mmol/L) was
added into Eppendorf cups and placed under a cell culture flow bank for two hours. After the
evaporation of the methanol, HDL was added at the indicated concentration and amounts, vigorously
vortexed and left at room temperature for one hour. Loading of HDL by this method led to nearly total
uptake of the provided S1P as determined by LCMS. Same HDL-S1P values after loading were the
same with and without subsequent dialysis demonstrating that HDL stably retained all of the acquired
S1P.
Injection of C17-S1P–loaded erythrocytes
Erythrocytes were isolated from blood of C57Bl/6 mice obtained by cardiac puncture as described
above. C17-S1P was added to a final concentration of 10μmol/L to the erythrocyte suspension
(hematocrit of 0.5) for 1 hour at 37°C (6). After several washes, 150μl of the suspension were injected
into the retroorbital plexus of each mouse. Mice were sacrificed 5 minutes later.
Mice
Mice heterozygous for HNF 1A (7) were a gift from the Institut Pasteur, Paris. C57Bl/6 mice were
from the central animal laboratory of the University Hospital Essen. 4-Deoxypyridoxine (DOP;
6mg/L) was administered with the drinking water for 16 days. Injections were performed on
3
anesthetized animals (isoflurane). The study was approved by the Landesamt für Natur, Umwelt und
Verbraucherschutz Nordrhein-Westfalen.
Cell culture experiments
Chinese hamster ovarian cells (CHO-K1), CHO cells stably transfected with the human S1P1 receptor
(CHO-S1P1, both from Novartis) or human umbilical vein endothelial cells (HUVEC) were cultured at
37°C, 5% CO2, in the corresponding media (CHO-K1: F-12 (Ham’s)+GlutaMAX, supplemented with
10% fetal calf serum (FCS), % penicillin/streptomycin/amphotericin B (PSA); CHO-S1P1:
aMEM+GlutaMAX, 10% FCS, 1% L-glutamate, 1% PSA, 50μg/ml gentamicin, and 0.5mg/mL G418;
HUVEC: RPMI 1640, 20% FCS, 1% PSA, 1% heparin, 0.5% bovine pituitary extract; all cell culture
reagents were purchased from Gibco/Invitrogen. Confluent cells were serum-starved overnight (CHO)
or for four hours (HUVEC) in serum-free medium prior to incubation with different concentrations of
isolated HDL for the indicated durations. The S1P1-receptor antagonists W146 (Avanti Polar Lipids)
and NIBr (Novartis), and the S1P3-receptor antagonist TY52156 (8) synthesized and characterized by
us previously (9) were used as indicated. In some experiments, HDL-bound S1P was blocked by
incubating HDL with different concentrations of Sphingomab (a kind gift of R. Sabbadini, LT1002,
Lpath) for 30 minutes at 37°C prior to adding them to the cells. After stimulation, cells were washed
once in ice-cold PBS and scraped in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM
MgCl2, 5 mM EDTA, 5 mM EGTA, 10 mM NaF, 10% glycerol, 0.5% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 mM NaVO4, 1 µg/ml aprotinin, leupeptin, and pepstatin) for 15 min
on ice. After centrifugation at 15,000xg for 5 min, the supernatants were taken and protein
concentrations determined using the BCA protein assay (Pierce). For functional experiments, a total of
HDL preparations from 14 healthy individuals and 9 patients with stable coronary artery disease
(sCAD) were randomly chosen from the human HDL sample pool. Demographic and clinical data of
these subjects and patients, respectively, are provided in Supplemental Table 2.
4
Immunoblotting
Cell lysates (20μg of protein) or HDLs (10μg) were denatured with sample buffer (0.5mol/L TrisHCl,
pH 6.8, 10% Glycerol, 10% SDS, 5% b-mercapto-ethanol, 0.1% bromphenol blue) and boiled for 10
minutes. Samples were separated in SDS-polyacrylamide gels and transferred onto Immobilon
polyvinylidene difluoride membranes (Millipore). Immunoblotting was performed by incubating the
PVDF membranes with primary antibodies against pERK1/2 (1:1000), pAkt (1:500) or peNOS (Ser
1177) (1:100) or the respective total proteins ERK1/2 (1:2000), Akt (1:250) or eNOS (1:100)
overnight at 4°C. All antibodies were from Cell Signaling Technologies). Western blotting on HDL
samples was performed using antibodies to apoM (Santa Cruz goat polyclonal anti-apoM, 1:200, or
Abnova mouse monoclonal anti-apoM, 1:50) or to apoAI (Abcam mouse monoclonal anti-apoAI,
1:1000) for 1 hour at 37°C. Secondary antibodies were HRP-conjugated (against rabbit or mouse,
Vector, Santa Cruz) and were used at a dilution of 1:5000 at room temperature for 1 hour. Signals
were detected using electrochemiluminescence (Amersham ECL Western Blotting detection reagents,
GE) and analyzed/quantified with Image Lab (BioRad).
Vasodilation studies
Vasodilation studies with HDLs were performed on norepinephrine-precontracted rat mesenteric
arteries as previously published (10) and following established protocols (11). Male Lewis rats were
sacrificed after enflurane-inhalation anesthesia by rapidly removing the heart in accordance with the
German laws for animal welfare and with approval by the local review committee. Segments of 2 mm
length of rat mesenteric arteries with intact endothelium were mounted into a Mulvany myograph
(Danish Myo Technology, Aarhus, Denmark) and equilibrated with Krebs-Henseleit buffer. Maximal
vasoconstriction was defined by the response to potassium chloride (KCl; 1.2*10 -1 mol/L).
Subsequently, arteries were washed and re-challenged with norepinephrine (10-5 mol/L) and carbachol
(10-4 mol/L) to verify a strong agonist response and endothelial functionality. Endothelial integrity was
defined as vasodilation to carbachol by 80% of the norepinephrine-induced preconstriction
amplitude. The dilator responses of mesenteric arteries to S1P-loaded or native HDL were determined
after preconstriction by 10-5 mol/l norepinephrine. When maximal vasoconstriction had been reached,
5
dilation was induced by adding cumulatively increasing concentrations of native or S1P-loaded HDL.
Dilator responses were expressed as percent of the maximum vasoconstriction induced by
norepinephrine.
Statistics
Data are expressed by mean±standard deviation or median (range) for continuous variables, and
frequency count and percentage for qualitative variables, respectively. Groups were compared by
unpaired or paired t-test, Mann-Whitney-Wilcoxon-U-test or Chi2-test. Vasoconstrictor responses to
native and S1P-loaded HDL were compared using 2-way repeated measures ANOVA followed by
Bonferroni’s post-hoc tests. P-values are understood to be strictly descriptive. Statistical significance
was assumed for P<0.05. Analyses and graphs were performed with Microsoft Office Excel or PASW
Statistics 18.0 or 19.0 (Chicago, USA).
6
Supplemental figures
—
1
CHO-K1
CHO-S1P1
0.1
0.1
2.5
5
10
—
1
2.5
S1P [µM]
5
10
S1P incubation time [min]
pERK1/2
Supplemental Figure 1.
Comparison of S1P signaling in CHO-K1 and CHO-S1P1 cells.
CHO-K1 control cells and CHO cells overexpressing the human S1P1
receptor (CHO-S1P1) were stimulated with 0.1 µM S1P for the
indicated times and ERK1/2 phosphorylation determined by Western
blotting.
7
ApoM in HDL/apoM in reference HDL
4.0
P=0.26
3.0
2.0
1.0
0
Controls
Supplemental Figure 2.
sCAD
The HDL content of apoM is similar in healthy and CAD-HDL.
The HDL content of apolipoprotein M was determined by Western
blotting in HDL (2.5μg of protein) isolated from 70 control subjects
and 64 patients with stable coronary artery disease (sCAD).
Demographic and clinical data of the subjects are provided in
Supplemental Table 1. The HDL sample of one independent, healthy
subject was loaded on each SDS-gel to serve as a reference sample.
The signal intensity of each apoM band (in arbitrary units) was
expressed in relation (ratio) to the reference sample ran on the same
gel (in arbitrary units). Densitometry was performed with ImageLab
Software (BioRad Laboratory; values for the ratio in healthy HDL
(0.86 [0.27-2.49]), values for the ration in CAD-HDL (0.95 [0.223.31]; P=0.26). Box plots show median, minimum and maximum.
Circles indicate outliers (values above or below 1.5 times the
interquartile range); asterisks indicate extreme values (values above or
below 3 times the interquartile range).
8
Supplemental Table 1. Demographic and clinical data of all study participants.
Controls
Stable CAD
(n=70)
(n=64)
41 (59)
43 (67)
n.s.
51 (20 – 84)
66 (37 – 86)
< 0.05
Total cholesterol [mmol/L]
5.68 (3.53 – 7.48)
4.15 (2.79 – 8.93)
< 0.05
HDL-Cholesterol [mmol/L]
1.57 (0.75 – 2.94)
1.23 (0.65 – 2.27)
< 0.05
LDL-Cholesterol [mmol/L]
3.15 (1.47 – 4.62)
2.24 (1.34 – 5.47)
< 0.05
LDL-C:HDL-C ratio
1.96 (0.70 – 3.83)
1.93 (0.83 – 4.16)
n.s.
n.a.
59 (95)
45 (64)
63 (98)
n.a.
18 (28)
Male [no. (%)]
Age [years]
Statins [no. (%)]
Hypercholesterolemia [no. (%)]
Diabetes mellitus [no. (%)]
P-value
< 0.05
9
Severity of symptoms [no. (%)]
- CCS I
n.a.
34 (53)
- CCS II
24 (38)
- CCS III
6 (9)
- CCS IV
0
Extent of disease [no. (%)]
- 1-vessel-disease
- 2-vessel-disease
17 (27)
n.a.
17 (27)
- 3-vessel-disease
30 (47)
Previous PCI [no. (%)]
n.a.
52 (84)
Coronary artery bypass graft [no. (%)]
n.a.
26 (41)
BMI – body mass index, CAD – coronary artery disease, HDL-Cholesterol – high density-lipoprotein-cholesterol, LDL-Cholesterol – low densitylipoprotein-cholesterol, n.a. – not available/not applicable, n.s. – not significant, PCI – percutaneous coronary intervention. Patients were classified as
hypercholesterolemic if the diagnosis appeared in the patients’ file regardless of current lipid levels, or if current plasma total cholesterol was > 5.16
mmol/L. CAD patients have lower LDL-C most probably due to their statin medication. Data are presented as mean (minimum – maximum) or as
number and percentage.
10
Supplemental Table 2. Demographic and clinical data of all subjects whose HDL were used for functional experiments.
Healthy individuals
Stable CAD patients
(n=14)
(n=9)
4 (29)
6 (67)
n.s.
35 (20 – 67)
69 (50 – 75)
< 0.05
Total cholesterol [mmol/L]
6.24 (3.69 – 6.81)
4.26 (3.46 – 5.44)
< 0.05
HDL-Cholesterol [mmol/L]
2.0 (1.06 – 2.37)
1.32 (0.65 – 1.70)
< 0.05
LDL-Cholesterol [mmol/L]
3.26 (1.73 – 4.05)
2.13 (1.68 – 3.17)
< 0.05
LDL-C:HDL-C ratio
1.6 (0.93 – 2.84)
1.78 (1.06 – 3.15)
n.s.
n.a.
9 (100)
n.a.
7 (70)
9 (100)
n.a.
n.a.
6 (67)
n.a.
Male [no. (%)]
Age [years]
Statins [no. (%)]
Hypercholesterolemia [no. (%)]
Diabetes mellitus [no. (%)]
P-value
11
Severity of symptoms [no. (%)]
- CCS I
n.a.
5 (56)
- CCS II
4 (44)
- CCS III
0
- CCS IV
0
Extent of disease [no. (%)]
- 1-vessel-disease
- 2-vessel-disease
3 (33)
n.a.
6 (67)
- 3-vessel-disease
0
Previous PCI [no. (%)]
n.a.
4 (44)
Coronary artery bypass graft [no. (%)]
n.a.
3 (33)
BMI – body mass index, CAD – coronary artery disease, HDL-Cholesterol – high density-lipoprotein-cholesterol, LDL-Cholesterol – low density-lipoproteincholesterol, n.a. – not available/not applicable, n.s. – not significant, PCI – percutaneous coronary intervention. Patients were classified as having
hypercholesterolemia if the diagnosis once appeared in the patients’ file regardless of current lipid levels, or if current plasma total cholesterol was > 5.16
mmol/L. Data are presented as mean (minimum – maximum) or as number and percentage.
12
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