Online Supplementary Materials
Oxidized Phospholipids, Lipoprotein(a) and Progression
Of Calcific Aortic Valve Stenosis
AUTHORS: Romain Capoulade, PhD, Kwan L. Chan, MD, FRCPC, Calvin Yeang, MD,
PhD, Patrick Mathieu, MD, FRCSC, Yohan Bossé, PhD, Jean G. Dumesnil, MD,
FRCPC, FACC, James W. Tam, MD, FRCPC, Koon K. Teo, MBBCh, PhD, FACC,
Ablajan Mahmut, MD, MSc, Xiaohong Yang BSc, Joseph L. Witztum, MD, Benoit J.
Arsenault PhD, Jean-Pierre Després, PhD, FAHA, Philippe Pibarot, DVM, PhD, FACC,
FAHA, FESC, FASE, Sotirios Tsimikas, MD, FACC
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Online Supplementary Methods
Clinical data
Clinical data included age, gender, height, weight, waist circumference, body
surface area (BSA), body mass index (BMI), systolic and diastolic blood pressures,
hypertension (patients receiving antihypertensive medications or having known but
untreated hypertension [blood pressure ≥ 140/90 mm Hg]), history of smoking and
randomization status (i.e. statin vs placebo). The clinical identification of patients with
the features of the metabolic syndrome (MetS) was based on the modified criteria
proposed by the National Cholesterol Education Program – Adult Treatment Panel III
(1).
Doppler echocardiographic data
The sonographers at each site received training in the acquisition and
interpretation of the echocardiograms before the beginning of the study. Randomly
selected studies that contributed 10% of the total number of echocardiograms were
reviewed to ensure that the studies and measurements were performed in accordance
with the protocol.
Aortic valve morphology and function: The aortic valve (AV) phenotype (i.e.
bicuspid vs. tricuspid) was recorded. The Doppler echocardiographic indices of AS
severity included peak aortic jet velocity (Vpeak), peak and mean transvalvular pressure
gradients obtained with the use of the modified Bernoulli equation, and the aortic valve
area (AVA) calculated by the standard continuity equation. The AVA could not be
determined in 16% (n=35) of the patients due to subvalvular flow acceleration or
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inadequate measure of LV outflow tract diameter. The degree of AV calcification was
scored according to the criteria proposed by Rosenhek et al (2).
Left ventricular geometry and function: LV ejection fraction (LVEF) was measured
with the use of biplane Simpson method. The relative wall thickness ratio was
calculated by dividing the sum of the LV posterior wall and inter-ventricular septal
thicknesses by the LV internal dimension (3). Left ventricular mass was calculated with
the corrected formula of the American Society of Echocardiography and was indexed to
a 2.7 power of height (3, 4). As a measure of global LV hemodynamic load, we
calculated the valvulo-arterial impedance (5): Zva=(SBP+ΔPmean)/SVi where SBP is the
systolic blood pressure, ∆Pmean the mean transvalvular gradient, and SVi is the stroke
volume indexed to a 2.04 power of height (4).
Measurement of OxPL-apoB, OxPL-apo(a) and Lp(a) levels
OxPL-apoB levels were measured with a chemiluminescent immunoassay using
the murine monoclonal antibody E06 that recognizes the phosphocholine (PC) group on
oxidized but not on native phospholipids (6-10). E06 similarly recognizes the PC
covalently bound to bovine serum albumin (BSA) in PC-BSA. A 1:50 dilution of plasma
in 1% BSA in TBS was added to microtiter wells coated with the apoB-100 specific
monoclonal antibody MB47, which binds a saturating amount of apoB-100 to each well,
and biotinylated E06 was then added to determine the content of OxPL-apoB. These
values are reported as nanomolar (nM) PC-OxPL using a standard curve of nM PC
equivalents, as recently described (11). Because each well contains equal numbers of
apoB-100 particles, the OxPL-apoB value reflects the absolute content of OxPL per a
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constant amount of captured apoB lipoprotein. It thus represents an OxPL-apoB value
that is independent of plasma levels of apoB-100 or LDL cholesterol. Furthermore, the
assay detects only the subset of OxPL detected by antibody E06 and not all species of
OxPL. In prior studies, this variable was expressed as OxPL/apoB, reflecting the fact
that this measure quantitates the number of OxPL moles per unit mass of apolipoprotein
B-100 present on microtiter well plates (and not the level in the circulation). The
nomenclature is now changed to OxPL-apoB to minimize confusion that this measure
represents a ratio of OxPL divided by plasma levels of apoB. Within-person 5-year
reproducibility of frozen samples has been shown to be high (r=0.78) (12) and pilot-tests
showed that OxPL-apoB levels are stable over 24 hours on ice (intraclass correlation
coefficient 0.96) (11) as well as frozen samples stored under long term conditions (1315). OxPL-apo(a) levels were measured in an analogous manner to OxPL-apoB, except
that the capture antibody, LPA4 (16), which detects apolipoprotein(a) was used to
capture apo(a). The values are also reported in nM. Because these assays gave similar
results to OxPL-apoB, the OxPL-apo(a) data are only shown in the Supplement. Please
note that the absolute amounts of apoB-100 and apo(a) captured on the wells in the
respective assays are different and therefore one can not directly compare the absolute
OxPL-apoB and OxPL-apo(a) values.
Lp(a) levels were measured by a validated chemiluminescent ELISA as
previously described (6).
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Online Supplementary Results
Progression rate of aortic valve area
The results of the analysis of the progression rate of AVA (i.e. annualized AVA
progression rate) were strongly similar to those reported for the progression rate of
Vpeak.
Patients in the top tertile of Lp(a) had significantly faster progression of AVA
compared to those in the middle and bottom tertiles (-0.08±0.11 vs. -0.04±0.12 cm²/yr,
p=0.03). Patients in the top tertile of OxPL-apoB also displayed significantly faster
stenosis progression than those in the middle and bottom tertiles (-0.09±0.10 vs. 0.04±0.12 cm²/yr, p=0.01). After multivariable analysis, using similar models as for Vpeak,
top tertiles of Lp(a) or OxPL-apoB remained independently associated with AVA
progression rate (all p<0.05).
In patients ≤57 years, the results were also highly consistent with those reported
with Vpeak: patients in the top tertile of Lp(a) or OxPL-apoB had faster stenosis
progression (annualized AVA: -0.10±0.13 vs. -0.02±0.12 cm²/yr, p=0.003; -0.10±0.13
vs. -0.02±0.12 cm²/yr, p=0.002; respectively), and these relationships remained
significant after multivariable adjustment (all p<0.05).
Relationship of OxPL-apo(a) with AS progression rate and clinical outcomes
The relationship between OxPL-apo(a) and AS progression rate or clinical
outcomes were similar to those obtained with OxPL-apoB. In the overall group, median
OxPL-apo(a) levels were 15.7 [4.5-51.7] nM and OxPL-apo(a) levels were strongly
correlated to OxPL-apoB levels (r=0.86; p<0.001). Patients in the top tertile of OxPL-
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apo(a) (>33.5 nM) had significantly faster stenosis progression compared to those in the
middle and bottom tertiles (+0.26±0.26 vs. +0.17±0.21 m/s/yr, p=0.005). After
multivariable analysis, using similar models as for OxPL-apoB (Multivariable Model #1,
Table 2), OxPL-apo(a) remained independent predictors of faster progression rate of
Vpeak (top tertile of OxPL-apo(a): βeta coeff.=0.22±0.04, p=0.02). Patients in the top
tertile of OxPL-apo(a) had a higher risk of being rapid progressors in univariable
(OR=2.1; 95% CI 1.2-3.8; p=0.009) and multivariable (OR=2.8; 95% CI 1.4-5.4;
p=0.002) analysis.
There was a significant interaction between patient’s age and OxPL-apo(a)
(p=0.05) for AS progression. Among patients with median age ≤57 years, AS
progression was 2-fold faster in those in the upper tertile of OxPL-apo(a) (>33.5 nM)
compared to middle and bottom tertiles (+0.26±0.32 vs. +0.13±0.17 m/s/yr, p=0.005).
After multivariable adjustment, using similar models as for OxPL-apoB, OxPL-apo(a)
remained a powerful and independent predictor of faster AS progression rate in patients
≤57 years (all p≤0.01). In the subset of patients ≤57 years, those in the top tertile of
OxPL-apo(a) had a higher risk of being rapid progressors in univariable (OR=4.0; 95%
CI 1.7-9.5; p=0.001) and multivariable (OR=5.6; 95% CI 1.8-16.8; p=0.002) analysis.
After adjustment for age, male gender and baseline AS severity, patients in the
top tertile of OxPL-apo(a) had a significantly higher risk of clinical events, in the overall
group (HR: 1.8, 95% CI 1.0-3.3, p=0.049) and in the subset of patients ≤57 years (HR:
4.2, 95% CI 1.3-13.4, p=0.01).
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Online Supplementary Figure
ONLINE FIGURE S1: Impact of Aortic Valve Phenotype and Randomization Status on
AS Progression Rate according to Plasma Levels of Lp(a) and OxPL-apoB.
Caption: Comparison of annualized progression rate of peak aortic jet velocity after
dichotomization by aortic valve phenotype (i.e. bicuspid vs. tricuspid; Panels A and B) or
randomization status (i.e. statin vs. placebo; Panels C and D) in patients in the middle
and bottom tertiles vs. top tertile of Lp(a) (i.e. Lp(a) ≤ vs. >58.5 mg/dL; Panels A and C)
and OxPL-apoB (i.e. OxPL-apoB ≤ vs. >5.50 nM; Panels B and D).
ApoB: apolipoprotein B; Lp(a): lipoprotein(a); OxPL: oxidized phospholipids; Vpeak: peak
aortic jet velocity. ¶: p<0.05 compared to “Lp(a)≤50-Placebo” group; #: p<0.05 compared
to “Tertiles 1 & 2 of OxPL-apoB-Placebo” group. Error bars represent the SEM.
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Investigators and Sites of the ASTRONOMER trial
Kwan Leung Chan, Ian Burwash, University of Ottawa Heart Institute (Ottawa, ON)
Susan Fagan, Bruce Sussex, HSC-General Hospital (St. John’s, NFLD)
Chris Koilpillai, Bruce Josephson, QEII HSC (Halifax, NS)
Jean G. Dumesnil, Philippe Pibarot, Quebec Heart Institute (Laval, QC)
George Honos, Jewish General Hospital (Montreal, QC)
Jean-Claude Tardif, Montreal Heart Institute (Montreal, QC)
Michele Turek, The Ottawa Hospital – General Campus (Ottawa, ON)
Trevor Robinson, Chi-Ming Chow, St. Michael’s Hospital (Toronto, ON)
Sam Siu, Eric Yu; Toronto General Hospital (Toronto, ON)
Cam Joyner, Chris Morgan, Sunnybrook HSC (Toronto, ON)
Charles Tomlinson, Koon Teo, Hamilton HSC (Hamilton, ON)
James Tam, David Mymin, Nasir Shaikh, Davinder Jassal, HSC Winnipeg (Winnipeg)
Bibiana Cujec, University of Alberta Hospital (Edmonton, AB)
Peter Giannoccaro, Brian Connelly, Peter Lougheed Centre (Calgary, AB)
Kenneth O’Reilly, William Hui, Ted Fenske, Alan Jones, Randy Williams (Royal
Alexander Hospital (Edmonton, AB)
John Jue, Ken Gin, Pui-Kee Lee, Vancouver General Hospital (Vancouver, BC)
Randall Sochowski, Dennis Morgan, Mangeet Mann, Ken Yvorchuk, Victoria Heart
Institute (Victoria, BC)
Stephen Pearce, S. Cheung, Raymond Dong, J.M. Kornder, Dante Manyari, Salima
Shariff, Surrey Memorial Hospital (Surrey, BC)
Stuart Smith, Clause Rinne, Westheights Cardiology Clinic (Kitchener, ON)
Thao Huynh, Montreal General Hospital (Montreal, QC)
Milan Gupta, David Borts, Anil Gupta, Nickie Bonafede, Brampton Research Associates
(Brampton, ON)
Shekhar Pandey, Cambridge Cardiac Care Centre (Cambridge, ON)
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