The plasma concentration of Lpa-I:A-II particles as a predictor of the

Atherosclerosis 202 (2009) 304–311
The plasma concentration of Lpa-I:A-II particles as a predictor of the
inflammatory response in patients with ST-elevation
myocardial infarction
Monica Gomaraschi a , Gianfranco Sinagra b , Laura Vitali Serdoz b , Cristina Pitzorno b ,
Maurizio Fonda c , Luigi Cattin c , Laura Calabresi a , Guido Franceschini a,∗
a
b
c
Center E. Grossi Paoletti, Department of Pharmacological Sciences, University of Milano, Italy
Intensive Care Unit, Cardiovascular Department, Ospedali Riuniti and University of Trieste, Italy
Metabolic Unit, Department of Internal Medicine, Ospedali Riuniti and University of Trieste, Italy
Received 31 January 2008; received in revised form 2 April 2008; accepted 5 April 2008
Available online 12 April 2008
Abstract
Objective: To investigate the relationship between plasma HDL at admission and the extent of the inflammatory response during an ST-elevation
myocardial infarction (STEMI), and to analyse structural HDL changes during STEMI as related to the extent of inflammation.
Methods and results: CRP and IL-6 were monitored for 96 h in 45 patients with STEMI. Plasma apoA-II and LpA-I:A-II levels at admission,
but not HDL cholesterol or other HDL-related biomarkers, were associated with the extent of the inflammatory response during STEMI, as
indicated by the positive correlations with CRP AUC (apoA-II: F = 7.44, p = 0.009; LpA-I:A-II: F = 14.29, p < 0.001), and IL-6 AUC (apoAII: F = 6.98, p = 0.012; LpA-I:A-II: F = 6.67, p = 0.013). By multivariate analysis the plasma LpA-I:A-II level at admission was a powerful
independent predictor of the inflammatory response, evaluated either as CRP AUC (F = 22.30, p < 0.001), or IL-6 AUC (F = 6.92, p = 0.012).
During STEMI, the plasma concentration of LpA-I:A-II, but not LpA-I particles decreased, HDL became larger and progressively enriched
in serum amyloid A; these changes occurred only in patients with a significant inflammatory response.
Conclusion: An elevated plasma concentration of LpA-I:A-II particles was an independent predictor of a more severe inflammatory response
in patients with STEMI.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Myocardial infarction; High-density lipoproteins; Lipoprotein subpopulations; Inflammation; Acute phase response
1. Introduction
Inflammatory biomarkers, such as C-reactive protein
(CRP), interleukin-6 (IL-6), and serum amyloid A (SAA)
are key components of the acute phase response (APR)
to myocardial injury. During an acute coronary syndrome
(ACS), the APR is characterized by a rapid increase in
the plasma levels of these biomarkers, that triggers further
systemic and local inflammation. A high degree of interindividual variation in APR has been described in patients
with ACS, in whom a greater APR, as indicated by an ele∗
Corresponding author. Tel.: +39 0250319911; fax: +39 0250319900.
E-mail address: [email protected] (G. Franceschini).
0021-9150/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.atherosclerosis.2008.04.004
vated IL-6 or CRP level, is associated with a complicated
in-hospital course and an enhanced 14-day mortality [1,2].
Several prospective studies have clearly established that
plasma high-density lipoprotein (HDL) cholesterol levels are
inversely related to the incidence of acute coronary events
[3]. Besides being a strong independent predictor of the
occurrence of primary coronary events, a low plasma HDL
cholesterol level is also associated with unfavorable prognosis in patients who have recovered from a myocardial
infarction [4,5]. Whether this association reflects accelerated atherogenesis, or a direct detrimental effect of low HDL
on postischemic myocardial function is unknown. Recent
experimental and clinical evidence indicates that the antiinflammatory properties of HDL may be as important as
M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
their cholesterol transport function for cardiovascular protection [6,7]. Notably, in response to an inflammatory stimulus,
HDL can undergo complex structural changes, becoming less
functional or frankly proinflammatory [8]. It remains to be
established whether HDL exert anti-inflammatory effects in
the course of an ACS, and whether inflammation during an
ACS affects HDL structure. Therefore, a series of patients
with ongoing ACS was monitored: (i) to investigate the relationship between plasma content of HDL, apolipoproteins,
and subpopulations at admission, and the extent of the inflammatory response during ACS; and (ii) to analyse structural
HDL changes during ACS as related to the extent of inflammation.
2. Methods
2.1. Patients
Forty-five patients presenting with ST-elevation myocardial infarction (STEMI) between March–December 2002 and
July–December 2003 were enrolled in the study. They were
selected from a cohort of 160 patients consecutively admitted
to the Intensive Care Unit of the Ospedali Riuniti in Trieste
with a diagnosis of STEMI, defined using the standard criteria. 115 patients were not enrolled because of (i) admission
>6 h from the onset of symptoms (n = 89); (ii) lack of consent (n = 15); (iii) Killip class >3 at admission (n = 6); and (iv)
inadequate blood sampling (n = 5). A detailed clinical history
was collected for each subject. The study was approved by the
local institutional Ethic Committee, and all enrolled patients
gave written informed consent for participation in the study.
305
(LpA-I:A-II) was determined by electroimmunodiffusion
(Sebia Italia, Firenze, Italy) [9]. HDL protein composition
was assessed by SDS-PAGE, followed by Coomassie Blue
R250 staining, and quantitation of protein bands by densitometry. HDL particle size and subclass distribution were
determined by non-denaturing polyacrylamide gradient gel
electrophoresis (GGE) [9]; the relative protein content of
HDL subclasses was determined by dividing the HDL profile
into three size intervals, small HDL (diameter 7.2–8.2 nm),
medium HDL (diameter 8.2–8.8 nm) and large HDL (diameter 8.8–12.7 nm) [9].
2.3. Statistical analyses
The extent of variation in lipid/lipoprotein and inflammatory parameters during STEMI was assessed by calculating:
(1) the area under the curve (AUC) described by the single
parameter vs time (between admission and discharge); and (2)
the maximal change in the single parameter (Dmax ) by subtracting the value at admission from the peak value achieved
between admission and discharge. Results are reported as
mean ± S.E.M. Group differences in continuous variables
were evaluated by one-way ANOVA, with post hoc analysis
by the Neuman–Keuls test. For categorical variables, group
differences were examined with the use of 2 × 2 contingency
tables and a χ2 test of significance. Simple and multivariate
regression analyses were performed to assess the correlation
between parameters. Variables considered as potential predictors for multivariable modeling were selected by stepwise
forward selection, with entry and retention in the model set at
a significance level of 0.05. Group differences or correlations
with a p value <0.05 were considered statistically significant.
2.2. Biochemical analyses
3. Results
Blood was collected at admission, after 4, 8, 12, 24, 48,
72, and 96 h, and at hospital discharge. Plasma and serum
were prepared by low-speed centrifugation. Serum creatine
kinase (CK), CK-myocardial band (CK-MB) and Troponin I
(TnI) levels were measured by commercially available kits.
Plasma CRP and IL-6 levels were measured by immunoturbidimetry on a Roche Diagnostics Cobas 400 Analyzer and
by ELISA (R&D Systems, Minneapolis, USA), respectively.
The SAA content of the plasma total lipoprotein fraction
(d < 1.21 g/ml) was assessed by Western Blot analysis with
an antibody against human SAA (BioSource International,
Camarillo, USA); lipoprotein SAA content is expressed as
arbitrary units, normalized by a reference sample applied on
each gel.
Plasma total and HDL cholesterol (TC and HDL-C), and
triglycerides (TG) were determined by standard enzymatic
techniques. LDL-C was calculated using the Friedewald’s
formula. Apolipoprotein A-I (apoA-I), A-II and B levels were determined by immunoturbidimetry. The plasma
level of lipoprotein particles containing only apoA-I (LpAI) and of particles containing both apoA-I and apoA-II
The characteristics of the enrolled patients are reported in
Table 1. Plasma lipids measured at admission were assumed
as pre-STEMI values (see Data Supplement). Twenty-one
out of the 45 enrolled patients suffered from unstable angina
before admission; their plasma lipid, CRP and IL-6 levels at
admission did not differ significantly from those of patients
without pre-STEMI angina.
3.1. Acute phase inﬂammatory response in STEMI
The plasma IL-6 level at admission ranged from 0.15 to
26.5 pg/ml (mean: 4.4 ± 0.7 pg/ml), and rapidly rose thereafter, reaching a maximum of 27.9 ± 3.9 pg/ml after 24 h
(Fig. 1). The plasma CRP level at admission ranged from
0.7 to 15.9 mg/l, remained stable for the first 8 h, and
then increased sharply (Fig. 1); the maximum CRP value
(35.2 ± 5.4 mg/l; range: 0–124 mg/l) was reached at 72 h.
Lipoprotein SAA was undetectable in all samples collected at
admission; it became detectable at 12–24 h, reaching a maximum signal at 48 h (Fig. 1). Both IL-6 and SAA returned to
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M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
Table 1
Clinical and biochemical features of STEMI patients at admission
Gender (M/F)
Age (y)
Pre-STEMI unstable angina (n)
30/15
62.8 ± 1.3 (45–80)
21
Cardiovascular risk factors
Hypoalphalipoproteinemia (HDL-C < 40 mg/dl)
Hypertension
Current or past smoke
Family history of myocardial infarction
Hypercholesterolemia (LDL-C > 160 mg/dl)
Diabetes
Obesity (body mass index > 30 kg/m2 )
71%
71%
71%
36%
27%
16%
13%
Pharmacological treatment at admission
ACE inhibitors or ␤-blockers
Aspirin
Statins
31%
20%
18%
Revascularization procedure
Thrombolytic therapy (n)
Primary PTCA (n)
43
2
Time from the onset of symptoms to admission (h)
CRP (mg/l)
IL-6 (pg/ml)
Total cholesterol (mg/dl)
LDL-cholesterol (mg/dl)
Triglycerides (mg/dl)
Apolipoprotein B (mg/dl)
HDL-cholesterol (mg/dl)
Apolipoprotein A-I (mg/dl)
Apolipoprotein A-II (mg/dl)
2.9 ± 0.2
2.3 ± 0.6
4.4 ± 0.7
211.8 ± 4.6
144.4 ± 4.6
149.8 ± 10.4
125.0 ± 4.4
37.4 ± 2.0
100.3 ± 3.9
29.5 ± 1.0
Data are expressed as mean ± S.E.M., n = 45.
Fig. 1. Acute phase inflammatory response in STEMI patients. Plasma IL6 (circles, pg/ml) and CRP (squares, mg/l) concentrations from admission
to discharge were measured by immunological methods, and reported as
means ± S.E.M. (n = 45). The SAA content of the total plasma lipoprotein fraction was assessed at the same time-points by western blotting; a
representative blot is shown at bottom.
baseline levels at discharge (on average 8 days after admission, range 5–14 days), when CRP was still significantly
elevated compared to admission (Fig. 1). No difference in the
extent of APR was found between patients with and without
pre-STEMI angina.
3.2. Plasma lipids and lipoproteins during STEMI
Changes in TC, LDL-C and TG levels are reported in the
Data Supplement and are consistent with previous findings.
Plasma HDL-C levels did not change during the first 72 h,
decreasing thereafter to an average value of 27.5 ± 2.0 mg/dl
at discharge (−26%; p = 0.003 vs admission) (Supplemental
Figure I).
No significant changes in plasma apoA-I levels were
detected during the observation period; in contrast, plasma
apoA-II levels rapidly declined after admission, with a minimum of 25.5 ± 0.9 mg/dl after 48 h (−14%; p = 0.004),
and remaining significantly lower than at admission until
discharge (−12%; p = 0.027) (Supplemental Figure I). Consistent with the variations in apoA-I and apoA-II levels, the
plasma LpA-I level did not change, whereas the LpA-I:A-II
level decreased to a minimum after 48 h (−21%, p = 0.004),
returning towards basal values thereafter (Supplemental
Figure II). No significant differences in plasma LpA-I:A-II
levels were observed between admission, discharge and 3 or
6 months follow-up.
3.3. Plasma lipids/lipoproteins at admission and the
extent of APR in STEMI
To understand the relationship between plasma
lipid/lipoprotein concentrations at admission and the
extent of APR in STEMI, two different analyses were
performed. First, patients were stratified according to the
peak CRP level and divided into two groups: those with a
peak CRP concentration below the median value of 37.5 mg/l
were defined as low-grade APR (low-APR) patients, and
those with a peak CRP concentration ≥37.5 mg/l were
defined as high-grade APR (high-APR) patients. Low-APR
patients presented at admission with lower plasma CRP
level than high-APR patients (Table 2). The greater plasma
CRP levels in high-APR patients were unrelated to the
prevalence of pre-STEMI angina. Plasma TC and LDL-C,
TG, apoB and LpA-I levels at admission were similar in the
two groups. In contrast, high-APR patients displayed greater
average plasma levels of HDL-C, HDL apolipoproteins and
LpA-I:A-II particles than low-APR patients, but only the
difference in apoA-II and LpA-I:A-II levels was significant
(Table 2). No significant differences were detected between
the two groups for plasma CK, CK-MB and TnI (Table 2).
The results of such analysis were similar when patients
were stratified according to the peak IL-6 concentration (not
shown).
In a second analysis, plasma lipid/lipoprotein levels measured at admission in all the enrolled patients were correlated
M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
307
Table 2
Clinical and biochemical features at admission of STEMI patients stratified according to the extent of acute phase response (APR) in STEMI
Gender (M/F)
Age (y)
Pre-STEMI unstable angina (n)
Smokers (%)
BMI (kg/m2 )
CK (U/l)
CK-MB (U/l)
TnI (ng/ml)
CRP (mg/l)
IL-6 (pg/ml)
Total cholesterol (mg/dl)
LDL-cholesterol (mg/dl)
Triglycerides (mg/dl)
Apolipoprotein B (mg/dl)
HDL-cholesterol (mg/dl)
Apolipoprotein A-I (mg/dl)
Apolipoprotein A-II (mg/dl)
LpA-I (mg/dl)
LpA-I:A-II (mg/dl)
Low-APR
High-APR
p
17/6
60.4 ± 1.9
13
65%
25.5 ± 0.7
1656 ± 302
193 ± 40
117 ± 27
0.6 ± 0.6
3.6 ± 0.6
211.7 ± 5.6
146.2 ± 5.3
156.5 ± 13.8
125.9 ± 5.5
34.1 ± 2.7
95.6 ± 5.6
27.5 ± 1.2
54.0 ± 3.1
40.8 ± 3.4
13/9
65.4 ± 1.6
8
77%
25.5 ± 0.7
1862 ± 203
224 ± 25
107 ± 21
4.0 ± 1.1
5.2 ± 1.2
211.9 ± 7.6
142.6 ± 7.8
142.7 ± 15.8
124 ± 7.5
40.8 ± 3.0
105.3 ± 5.4
31.6 ± 1.5
52.0 ± 3.8
53.4 ± 3.6
0.453
0.054
0.266
0.577
1.000
0.581
0.527
0.673
0.006
0.233
0.978
0.708
0.281
0.827
0.104
0.220
0.038
0.684
0.014
Data are expressed as mean ± S.E.M. p values are the result of one-way ANOVA or χ2 test of significance between low- and high-APR patients. p values < 0.05
are in italic.
with the extent of APR, as assessed by calculating the
AUCs for plasma CRP and IL-6 levels. Only plasma apoA-II
and LpA-I:A-II levels were associated with APR extent, as
indicated by the significant positive correlations with CRP
AUC (apoA-II: F = 7.44, p = 0.009; LpA-I:A-II: F = 14.29,
p < 0.001), and IL-6 AUC (apoA-II: F = 6.98, p = 0.012;
LpA-I:A-II: F = 6.67, p = 0.013). By multivariate analysis
including all variables listed in Table 2 only the plasma LpAI:A-II level at admission proved to be a powerful independent
predictor of APR extent, evaluated either as CRP AUC
(F = 22.30, p < 0.001), or IL-6 AUC (F = 6.92, p = 0.012).
3.4. APR and changes in plasma lipids/lipoproteins
during STEMI
To assess the relationship between the extent of APR
and changes in plasma lipids/lipoproteins during STEMI, the
individual plasma lipid/lipoprotein values at different timepoints were averaged for low-APR and high-APR patients
separately. Profiles for TC, LDL-C, TG, apoB, apoA-I, and
LpA-I were similar in low-APR and high-APR patients (not
shown). The late decrease in plasma HDL-C observed at discharge was significantly greater in high-APR than low-APR
patients (−14.2 ± 2.7 vs −7.6 ± 1.6 mg/dl, p = 0.038), consistent with the general assumption that plasma HDL-C levels
fall in acute inflammatory states [8]. Significant differences
were observed in the plasma apoA-II and LpA-I:A-II profiles
(Fig. 2). Low-APR patients showed non significant changes
in apoA-II and LpA-I:A-II levels, while the average apoAII and LpA-I:A-II levels in high-APR patients progressively
decreased after admission, reaching a minimum after 72 h
(−19%, p = 0.007 and −28%, p = 0.005, respectively), not
changing thereafter (Fig. 2). Average apoA-II and LpA-I:A-
II levels at discharge were similar in high-APR and low-APR
patients (Fig. 2).
The protein composition of the total HDL fraction isolated
from plasma of low-APR patients did not change during the
observation period, except for the appearance of a minor SAA
component between 24 and 72 h from admission, accounting
for a maximum 5% of total HDL protein content (Fig. 3).
By contrast, in high-APR patients, SAA became a significant HDL component already after 12 h from admission
and accounted on average for 14% (range 5.0–36.2%) of
total HDL protein at 48 h; HDL protein composition was
completely normalized at discharge (Fig. 3). The low-APR
and high-APR patients displayed a very close HDL particle
size distribution at admission (Supplemental Table I). Such
distribution did not change over time in low-APR patients;
by contrast, high-APR patients displayed a progressive shift
of the small–medium size HDL towards particles of larger
size (Fig. 4), with a progressive increase of the major HDL
component size (Supplemental Table I). Such changes were
maximal at 48–72 h, with a tendency towards baseline values at discharge. The temporal changes in HDL particle size
distribution paralleled the SAA enrichment of HDL, as indicated by the significant positive correlation between the Dmax
in HDL SAA content and the Dmax in the plasma content of
large HDL (F = 7.22, p = 0.015).
4. Discussion
The anti-inflammatory properties of HDL are best illustrated by the ability of plasma-derived HDL to inhibit the
expression of proinflammatory cytokines and cell adhesion
molecules in cultured endothelial cells [10,11], and the
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M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
Fig. 2. Plasma levels of apoA-II and LpA-I:A-II particles during STEMI
in low-APR (䊉) and high-APR patients (). Data are expressed as
mean ± S.E.M., n = 23 for low-APR and n = 22 for high-APR. *, significantly different from levels measured at admission (0 h); and #, significantly
different from low-APR patients at the same time-point.
capacity of synthetic HDL, usually containing apoA-I as
the sole protein component, to down-regulate the expression
of adhesion molecules and decrease neutrophil infiltration
in animal models of acute inflammation [12,13]. Whether
HDL also exert acute anti-inflammatory effects in the clinical setting remains to be established, although a genetically
determined low plasma HDL-C concentration in healthy
males has been recently associated with enhanced APR after
endotoxin challenge [14].
It is well recognized that plasma HDL are a highly
heterogeneous lipoprotein family consisting of several subpopulations with varying density, size and apolipoprotein
composition, and that at least some of the potentially atheroprotective functions of HDL relate to properties of specific
HDL subpopulations [15]. LpA-I and LpA-I:A-II particles
contain approximately 35% and 65% of total plasma apoA-I,
whereas virtually all apoA-II is in LpA-I:A-II [16]. LpA-I is
mainly found in HDL2 , while LpA-I:A-II particles predominate in HDL3 [17]. The role of LpA-I and LpA-I:A-II as risk
factors for CHD is controversial. In the Framingham Offspring study and in the VA-HIT study no association between
plasma LpA-I and/or LpA-I:A-II levels and CHD was found
[18], whereas in the PRIME study, the concentration of both
LpA-I and LpA-I:A-II particles was inversely related to the
incidence of CHD [19]. In none of these prospective studies a significant difference emerged in the anti-atherogenic
potential of the two subclasses. The presence of apoA-II was
reported to decrease, or have no influence on, cholesterol
efflux from cells [20–22], while the anti-inflammatory properties of isolated LpA-I and LpA-I:A-II particles have not
been investigated. The present study reveals that an elevated
plasma concentration of LpA-I:A-II particles is an independent predictor of a more severe APR during a STEMI,
raising the hypothesis of a lower anti-inflammatory, or even
a proinflammatory activity of these particles compared to
those containing apoA-I alone. The preferential interaction
of paraoxonase-1 (PON-1) and lecithin:cholesterol acyltransferase with LpA-I particles in human plasma [23,24] and
previous findings in apoA-II overexpressing mice appear to
support this hypothesis. Indeed, HDL isolated from transgenic mice overexpressing apoA-II, which have enhanced
plasma levels of human-like LpA-I:A-II particles [25], have
a pro-oxidant and proinflammatory activity dependent on the
degree of apoA-II expression and likely due to the displacement of PON-1 from HDL by apoA-II [26–28].
Earlier studies described significant structural and compositional alterations in HDL of patients suffering an acute
myocardial infarction: HDL isolated 2 days after the event
were larger and enriched in SAA, which accounted for as
much as 87% of the total HDL protein, as compared to HDL
from healthy individuals [29]. The present study extends
such previous observations by showing that these changes
are quantitatively and temporally related to the APR triggered by myocardial injury. By examining HDL particle size
and protein composition serially at admission and during
the course of the myocardial infarction, we could demonstrate that HDL becomes larger and enriched in SAA only
in high-APR patients. There is clear evidence that HDL
structure and metabolism is critically altered by the APR
driven by acute inflammation [8,29,30]. As a consequence,
the lipid and protein composition of circulating HDL is modified by a reduction in plasma levels and the conversion
from anti-inflammatory into proinflammatory particles [8].
Whether a subset of HDL particles is selectively affected
by APR is unknown. It has been reported that SAA associates preferentially with HDL3 [29], which include most
of LpA-I:A-II. Here we provide indirect evidence that LpAI:A-II particles are more sensitive to inflammation-induced
modifications, as a selective depletion of LpA-I:A-II particles was observed only in high-APR patients with the same
time-pattern of APR course; on the contrary LpA-I levels did
not change during myocardial infarction, even in high-APR
patients. The reasons of the oversensitivity of LpA-I:A-II particles to inflammation-induced modifications are presently
unknown.
M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
309
Fig. 3. HDL apolipoprotein composition during STEMI. Representative SDS-PAGE of the total HDL fraction collected from low-APR (panel A) and high-APR
(panel B) patients from admission (0 h) to discharge (D). Panel C, SAA HDL content (% of total protein) for low-APR (䊉) and high-APR patients (). Data
are expressed as mean ± S.E.M., n = 23 for low-APR patients and n = 22 for high-APR patients. *, significantly different from levels measured at admission
(0 h); and #, significantly different from low-APR patients at the same time-point.
Fig. 4. HDL particle size distribution during STEMI. Representative non-denaturing GGE of the d < 1.21 g/ml plasma fraction collected from low-APR (panel
A) and high-APR (panel C) patients from admission (0 h) to discharge (D). Average profiles of HDL particle size distribution at admission (grey dotted line)
and after 48 h (black continuous line) for low-APR (panel B) and high-APR (panel D) patients. n = 23 for low-APR patients and n = 22 for high-APR patients.
310
M. Gomaraschi et al. / Atherosclerosis 202 (2009) 304–311
4.1. Study limitations and conclusions
The present results suggest that an elevated plasma level
of LpA-I:A-II at the time of ACS may identify patients
with a more severe index event, but the sample size of
the present study was too small and the study was not
powered to obtain a reliable estimate of the role of LpAI:A-II in predicting outcome after a myocardial infarction.
Large prospective studies are needed to investigate the
prognostic significance of LpA-I:A-II levels and their utility for risk stratification in patients suffering an ACS.
All results achieved in the present study, including the
discovery of a relationship between basal LpA-I:A-II levels and the extent of APR induced by myocardial injury,
should be considered exploratory and hypothesis-generating.
Experimental studies are warranted to understand whether
HDL particles of distinct apolipoprotein composition may
differ in their anti-inflammatory and cardioprotective activities.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.atherosclerosis.
2008.04.004.
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