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Proinflammatory high-density lipoprotein as a biomarker for atherosclerosis in patients with systemic lupus erythematosus and rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 8, August 2006, pp 2541–2549
DOI 10.1002/art.21976
© 2006, American College of Rheumatology
Proinflammatory High-Density Lipoprotein as a Biomarker for
Atherosclerosis in Patients With Systemic Lupus Erythematosus
and Rheumatoid Arthritis
Maureen McMahon,1 Jennifer Grossman,1 John FitzGerald,1 Erika Dahlin-Lee,1
Daniel J. Wallace,2 Bernard Y. Thong,3 Humeira Badsha,3 Kenneth Kalunian,4
Christina Charles,1 Mohamad Navab,1 Alan M. Fogelman,1 and Bevra H. Hahn1
Results. SLE patients had more proinflammatory
HDL (mean ⴞ SD score 1.02 ⴞ 0.57, versus 0.68 ⴞ 0.28
in controls [P < 0.0001] and 0.81 ⴞ 0.22 in RA patients
[P ⴝ 0.001 versus SLE patients]). A higher proportion
of SLE patients had proinflammatory HDL: 44.7% of
SLE patients versus 4.1% of controls and 20.1% of RA
patients had scores >1.0 (P < 0.006 between all
groups). Levels of ox-LDL correlated with levels of
proinflammatory HDL (r ⴝ 0.37, P < 0.001). SLE
patients with CAD had significantly higher proinflammatory HDL scores than patients without CAD (P <
0.001).
Conclusion. HDLs are proinflammatory in a significant proportion of SLE patients and are associated
with elevated levels of ox-LDL. Abnormal HDLs impair
the ability to prevent LDL oxidation and may predispose to atherosclerosis.
Objective. Women with systemic lupus erythematosus (SLE) have a 7–50-fold increased risk of coronary
artery disease (CAD). In the general population, oxidized low-density lipoprotein (ox-LDL) increases the
risk for CAD. Normal high-density lipoproteins (HDLs)
protect LDL from oxidation; proinflammatory HDLs do
not. This study was undertaken to determine whether
patients with SLE, who have chronic inflammation that
causes oxidative damage, have more proinflammatory
HDL and higher levels of ox-LDL, thus predisposing
them to atherosclerosis.
Methods. One hundred fifty-four women with
SLE, 48 women with rheumatoid arthritis (RA), and 72
healthy controls were studied. The ability of the patients’ HDL to prevent oxidation of normal LDL was
measured. Values >1.0 (the value assigned for LDL
oxidation in the absence of HDL) after the addition of
HDL indicated proinflammatory HDL. Plasma ox-LDL
levels were measured as the amount of oxidation produced by the patient’s LDL after the removal of HDL.
Premature atherosclerosis is a major comorbid
condition in patients with systemic lupus erythematosus
(SLE). Young women with SLE have an estimated
50-fold increased risk of myocardial infarction compared
with age- and sex-matched controls (1). While SLE
patients are subject to the same traditional risk factors as
the general population (2–4), these factors do not
adequately account for the significantly increased level
of cardiovascular disease. Thus, there is a continuing
search for new biomarkers of increased risk for atherosclerotic disease in SLE patients. One possible biomarker is proinflammatory high-density lipoprotein (HDL).
Proinflammatory HDL is unable to perform its
usual protective role in the prevention of atherosclerosis. Atherosclerotic lesions begin when low-density lipoproteins (LDLs) are trapped in artery walls and are
seeded with reactive oxygen species (ROS) (5), resulting
Dr. McMahon’s work was supported by grants from the
American College of Rheumatology Research and Education Foundation and the Lupus Research Institute. Drs. Navab and Fogelman’s
work was supported by Bruin Pharmaceuticals. Dr. Hahn’s work was
supported by the Lupus Research Institute and by a Kirkland Award.
1
Maureen McMahon, MD, Jennifer Grossman, MD, John
FitzGerald, MD, PhD, Erika Dahlin-Lee, AB, Christina Charles, MD,
Mohamad Navab, PhD, Alan M. Fogelman, MD, Bevra H. Hahn, MD:
University of California at Los Angeles David Geffen School of
Medicine; 2Daniel J. Wallace, MD: Cedars Sinai Medical Center, Los
Angeles, California; 3Bernard Y. Thong, MD, Humeira Badsha, MD:
Tan Tock Seng Hospital, Singapore; 4Kenneth Kalunian, MD: University of California at San Diego, La Jolla.
Address correspondence and reprint requests to Maureen
McMahon, MD, Division of Rheumatology, UCLA David Geffen
School of Medicine, 1000 Veteran Avenue 32-59, Los Angeles, CA
90095. E-mail: mmcmahon@mednet.ucla.edu.
Submitted for publication August 26, 2005; accepted in
revised form April 13, 2006.
2541
2542
McMAHON ET AL
Table 1. Demographic and clinical data on the patients and controls*
Age, mean ⫾ SD years
Total cholesterol, mean ⫾ SD mg/dl
HDL, mean ⫾ SD mg/dl
LDL, mean ⫾ SD mg/dl
Triglycerides, mean ⫾ SD mg/dl
Ethnicity, no. (%)
Caucasian
Asian
African American
Hispanic
Ox-LDL, mean ⫾ SD FU‡
SLE patients
(n ⫽ 154)
Controls
(n ⫽ 72)
RA patients
(n ⫽ 48)
40.2 ⫾ 13.6
39.9 ⫾ 16.3
54.5 ⫾ 12.5
180.3 ⫾ 44.7
56.3 ⫾ 18.7
99.9 ⫾ 36.4
116.1 ⫾ 80.4
183.2 ⫾ 36.3
51.8 ⫾ 14.9
113.1 ⫾ 35.9
97.6 ⫾ 47.1
196.5 ⫾ 41.5
63.4 ⫾ 22.5
110.2 ⫾ 31.3
128.1 ⫾ 72.2
72 (46.7)
33 (21.4)
35 (48.6)
23 (31.9)
33 (68.8)
2 (4.2)
15 (9.7)
33 (21.4)
6,713.7 ⫾ 3,674.5
2.8 (2)
12 (16.7)
5,356.2 ⫾ 2,610.0
3 (6.3)
10 (20.8)
6,260.6 ⫾ 3,494.3
P†
⬍0.001 (controls vs. RA)
⬍0.001 (SLE vs. RA)
0.07 (SLE vs. RA)
0.007 (controls vs. RA)
0.04 (controls vs. SLE)
0.03 (controls vs. RA)
0.02 (SLE vs. RA)
0.001 (controls vs. RA)
0.001 (SLE vs. RA)
NS
NS
0.05 (controls vs. SLE)
* SLE ⫽ systemic lupus erythematosus; RA ⫽ rheumatoid arthritis; HDL ⫽ high-density lipoprotein; ox-LDL ⫽ oxidized low-density lipoprotein;
FU ⫽ fluorescence units.
† Only statistically significant P values are shown. NS ⫽ not significant.
‡ Determined in 83 patients with SLE, 39 healthy controls, and 28 patients with RA.
in oxidized LDL (ox-LDL) phospholipids (6). After
endothelial cells are exposed to these oxidized lipids,
they release cytokines, which induce monocyte binding,
chemotaxis, and differentiation of monocytes into macrophages (6). Ox-LDLs are phagocytized by infiltrating
macrophages, leading to the formation of foam cells, a
hallmark of atherosclerotic lesions (7).
Normal HDL removes ROS from LDL, preventing both the oxidation of LDL and the recruitment of
inflammation mediators (6,8). HDLs, however, are
“chameleon-like” lipoproteins (7) with the capacity to be
antiinflammation in the basal state and proinflammatory
during acute-phase responses. Antiinflammatory HDL
protects LDL from oxidation, while proinflammatory
HDL does not (7). Navab et al have developed a novel
assay that determines the functional properties of HDL
by evaluating the ability of a subject’s HDL to prevent
lipid oxidation (9,10). A study using this assay in nonSLE patients with coronary artery disease (CAD) and
normal lipid profiles revealed that 25 of 26 patients had
proinflammatory HDL (11). HDL function improved
with statin therapy, but not to normal levels (11).
The idea that proinflammatory HDL is a risk
factor for atherosclerosis in SLE is a novel hypothesis. In
this study, we set out to determine if levels of proinflammatory HDL differ between individuals with chronic
inflammatory conditions (SLE and rheumatoid arthritis
[RA]) and healthy controls. We also investigated
whether any traditional cardiac risk factors or diseasespecific factors are associated with the presence of
proinflammatory HDL. We hypothesize that patients
with SLE, who have ongoing oxidative damage due to
chronic inflammation, have proinflammatory HDL and
high levels of ox-LDL.
PATIENTS AND METHODS
Study population. Three groups of subjects were studied: pre-exclusion numbers were 161 SLE patients, 60 RA
patients, and 80 healthy controls. The lupus patients are
patients at the University of California, Los Angeles (UCLA),
Cedars Sinai Medical Center in Los Angeles, and Tan Tock
Seng Hospital in Singapore. All patients fulfilled at least 4 of
the 1997 revised American College of Rheumatology (ACR;
formerly, the American Rheumatism Association) classification criteria for SLE (12). The controls were women, healthy
by self-report, with no clinical manifestations of SLE. The RA
patients were patients attending UCLA clinics who met the
ACR criteria for RA (13). Patients were excluded if they had
taken any lipid-lowering agents within the previous 3 months.
After exclusions for statin use, the final group consisted of 154
SLE patients, 48 RA patients, and 72 controls. Clinical data
obtained on patients with SLE and RA included information
regarding disease state, cardiac risk factors, and medication
use (Tables 1 and 2). Because C-reactive protein (CRP)
measurements were not available for many patients, these data
are not included in the analysis. Subjects were included in the
study after providing written consent. The study was reviewed
and approved by the Institutional Review Board of UCLA.
Laboratory and clinical assessments. Blood samples
were collected and processed by sucrose cryopreservation, and
HDL was isolated from plasma as previously described (11).
Tests to measure plasma lipid concentrations, erythrocyte
sedimentation rate (ESR), serum complement levels, and
autoantibodies against DNA and cardiolipin were performed
in our clinical laboratory using standard methods. On the day
of serum sampling, the disease activity of SLE patients was
assessed using the Safety of Estrogens in Lupus Erythematosus: National Assessment (SELENA) version of the Systemic
INFLAMMATORY HDL AS A BIOMARKER FOR ATHEROSCLEROSIS IN SLE AND RA
Table 2.
2543
Disease characteristics and traditional cardiovascular risk factors in SLE and RA patients*
Characteristic
History of coronary artery disease, no. (%)†
History of cerebrovascular events, no. (%)‡
History of hypertension, no. (%)§
History of diabetes, no. (%)¶
Ever smoked, no. (%)
Currently smoking, no. (%)#
History of glomerulonephritis, no. (%)
Disease duration, mean ⫾ SD years
SELENA-SLEDAI score, mean ⫾ SD
SDI score, mean ⫾ SD
History of dsDNA antibody positivity, no. (%)
History of LAC positivity, no. (%)
History of aCL positivity (IgG, IgM, IgA), no. (%)
ANA positive, no. (%)
ESR, mean ⫾ SD mm/hour**
C3 level, mean ⫾ SD mg/dl
Current medications
Mycophenolate mofetil, no. (%)
Hydroxychloroquine, no. (%)
Cyclophosphamide, no. (%)
Methotrexate, no. (%)
Azathioprine, no. (%)
Anti-TNF␣ agents, no. (%)††
Current prednisone dosage, mean ⫾ SD mg/day
Cumulative lifetime prednisone dose, no. (%)
⬍10 gm
10–20 gm
⬎20 gm
SLE patients
(n ⫽ 154)
RA patients
(n ⫽ 48)
4 (2.7)
12 (7.8)
47 (30.5)
6 (3.9)
27 (17.5)
12 (7.8)
46 (30.0)
10.1 ⫾ 8.7
4.5 ⫾ 5.3
1.5 ⫾ 1.8
87 (56.5)
35 (22.7)
60 (38.9)
154 (100)
22.0 ⫾ 19.5
102.3 ⫾ 31.4
0
0
13 (27.1)
3 (6.3)
9 (18.8)
3 (6.3)
0
13.5 ⫾ 11.1
0
0
0
0
0
0
20.0 ⫾ 15.6
NA
27 (17.5)
89 (57.8)
9 (5.8)
7 (4.5)
21 (13.6)
0
7.85 ⫾ 15.8
0
7 (14.6)
0
25 (52.1)
3 (6.3)
30 (62.5)
3.17 ⫾ 9.04
80 (51.9)
25 (16.7)
45 (29.2)
36 (75)
4 (8.3)
7 (14.6)
* SLE ⫽ systemic lupus erythematosus; RA ⫽ rheumatoid arthritis; SELENA-SLEDAI ⫽ Safety of
Estrogens in Lupus Erythematosus: National Assessment version of the Systemic Lupus Erythematosus
Disease Activity Index; SDI ⫽ Systemic Lupus International Collaborating Clinics/American College of
Rheumatology Damage Index; dsDNA ⫽ double-stranded DNA; LAC ⫽ lupus anticoagulant; aCL ⫽
anticardiolipin antibody; ANA ⫽ antinuclear antibody; ESR ⫽ erythrocyte sedimentation rate; NA ⫽ not
available; anti-TNF␣ ⫽ anti–tumor necrosis factor ␣.
† Defined as a history of myocardial infarction, coronary artery disease documented on an angiogram or
stress test, or angina.
‡ Cerebrovascular events included transient ischemic attacks and stroke.
§ Defined as use of antihypertensive medication or systolic blood pressure ⬎140 mm Hg or diastolic blood
pressure ⬎90 mm Hg.
¶ Defined as a fasting glucose level ⱖ7.0 mmoles/liter (126 mg/dl), or treatment with insulin or an oral
hypoglycemic agent.
# Patients were defined as smokers if they had smoked any cigarettes within the previous 3 months.
** Data available for 142 SLE and 47 RA patients.
†† Adalimumab, etanercept, or infliximab.
Lupus Erythematosus Disease Activity Index (SLEDAI) (14).
Organ damage was determined using the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI) (15).
Cell-free assay. The cell-free assay is a modification of
a previously described method using LDL as the fluorescenceinducing agent (10). Normal HDL prevents oxidation of LDL
and therefore the oxidation of dichlorofluorescein (DCFH),
which releases a fluorochrome upon oxidation. To determine
the functional properties of HDL, the change in fluorescence
intensity resulting from oxidation of DCFH by LDL in the
presence or absence of test HDL was measured. LDL was
prepared from normal plasma as previously described (10).
Twenty microliters of the normal LDL solution (final concentration 50 ␮g/ml) and 90 ␮l of test HDL (at a final concentra-
tion of 10 ␮g/ml cholesterol) were incubated in 96-well plates
for 1 hour. Ten microliters of DCFH solution (0.2 mg/ml) was
then added to each well and incubated for 2 hours. Fluorescence was determined using a plate reader (Spectra Max,
Gemini XS; Molecular Devices, Sunnyvale, CA). Values of
DCFH activated by LDL in the absence of HDL (in fluorescence units [FU]) were normalized to 1.0 as the positive
control. Values ⬎1.0 FU after the addition of test HDL
indicated proinflammatory HDL; values ⬍1.0 indicated antiinflammatory HDL. Mean ⫾ SD values for intra- and interassay variability were 5.3 ⫾ 1.7% and 7.1 ⫾ 3.2%, respectively.
Oxidized LDL. Oxidized LDL was measured in an
unselected subset of 150 of the patients from all 3 groups, by
adding 200 ␮l of DCFH solution (0.2 mg/ml) to the apolipo-
2544
McMAHON ET AL
protein B (Apo B)/LDL–containing fraction of the patient’s
plasma. This solution (90 ␮l) was incubated for 2 hours in
96-well plates. Fluorescence was determined using a plate
reader. In addition, ox-LDL was measured in 38 SLE patients
or control subjects by sandwich enzyme-linked immunosorbent
assay (ELISA; Mercodia, Uppsala, Sweden), using the monoclonal antibody 4E6.
Statistical analysis. Results are expressed as the
mean ⫾ SD. Data were analyzed using SPSS 13.0 software
(SPSS, Chicago, IL). Skewed continuous variables were logarithmically transformed to attain a normal distribution. For
variables that did not attain a normal distribution by logarithmic transformation, nonparametric tests were used. Study
groups were compared using analysis of variance/Student’s
t-test for continuous variables and the chi-square test for
categorical variables. Correlation coefficients were calculated using simple regression, or Spearman’s rank correlation for abnormally distributed data. Logistic regression was
used to build models that identify risk factors associated
with the presence of proinflammatory HDL in SLE and RA
patients. P values less than 0.05 were considered significant.
RESULTS
Association of proinflammatory HDL with SLE
and RA. HDL samples were assessed for their pro- and
antiinflammatory properties using the cell-free assay.
HDL from SLE patients was more proinflammatory
than HDL from controls, with a mean ⫾ SD score of
1.02 ⫾ 0.57 in the SLE patients versus 0.68 ⫾ 0.28 in the
controls (P ⬍ 0.0001). HDL from RA patients was also
more inflammatory than HDL from controls, but less
than the HDL from SLE patients, with a mean ⫾ SD
score of 0.81 ⫾ 0.22 (P ⫽ 0.016 versus controls, P ⫽
0.001 versus SLE patients) (Figure 1A). Using a threshold of 1.0 as a cutoff to define proinflammatory HDL,
44.7% of SLE patients versus 20.1% of RA patients
versus 4.1% of controls had proinflammatory HDL (P ⬍
0.006 between all groups, P ⬍ 0.0001 between SLE
patients and controls) (Figure 1B).
Association of proinflammatory HDL with CAD.
Fourteen patients in the cohort, all in the SLE group,
had a history of documented clinical atherosclerosis.
Four patients had a history of CAD, 12 had a history of
cerebrovascular disease, and 2 patients had both. All 4
patients with CAD had proinflammatory HDL, with a
mean ⫾ SD score of 1.11 ⫾ 0.07, compared with 0.80 ⫾
0.43 in patients without CAD (P ⬍ 0.0001). Patients who
had had a stroke had a mean ⫾ SD score of 0.97 ⫾ 0.40,
versus 0.80 ⫾ 0.43 (P ⫽ 0.13) in patients who had not,
and half (6 of 12) had proinflammatory HDL. Overall,
the 14 patients with a documented history of atheroscle-
Figure 1. Association of proinflammatory high-density lipoprotein
(HDL) and systemic lupus erythematosus (SLE). A, Individual HDL
scores among subjects in the SLE, rheumatoid arthritis (RA), and
healthy control groups. Bars show the group means. P values were
determined by analysis of variance with Dunnett’s multiple comparison
test. B, Percentage of subjects with proinflammatory HDL. Note that
the proportion of subjects with proinflammatory HDL is highest in the
SLE group, but the proportion in the RA group is also increased
compared with controls. P values were determined by chi-square
analysis.
rosis had a mean ⫾ SD score of 1.01 ⫾ 0.33, versus
0.79 ⫾ 0.43 (P ⫽ 0.01) in patients without atherosclerosis, with 8 patients with atherosclerosis (57%) having
HDL in the proinflammatory range. In contrast, only
43.4% of the 140 patients without atherosclerotic events
had a score ⱖ1.0 (Figure 2A). Thus, in the SLE patients,
the presence of an atherosclerotic event increased the
likelihood of having proinflammatory HDL.
INFLAMMATORY HDL AS A BIOMARKER FOR ATHEROSCLEROSIS IN SLE AND RA
Figure 2. Association of HDL function and traditional and disease
risk factors. A, Association of HDL function and history of cardiovascular (CV) disease. Values are the mean and SD in 14 subjects. P
values were determined by Student’s t-test. B, Association of HDL
function and traditional risk factors for cardiac disease. Patients taking
lipid-lowering medications were excluded from this study. Values are
the mean and SD in 14 subjects. P values were determined by Student’s
t-test. C, Correlation of HDL function and levels of oxidized lowdensity lipoprotein (LDL), determined by Spearman’s rank test. Data
were analyzed in a subset of 83 SLE patients, 28 RA patients, and 39
healthy controls. D, Correlation of HDL function and erythrocyte
sedimentation rate (ESR; measured using the Westergren method), as
determined by Spearman’s rank test. CAD ⫽ coronary artery disease;
CVA ⫽ cerebrovascular accident; HTN ⫽ hypertension; FU ⫽
fluorescence units (see Figure 1 for other definitions).
Association of proinflammatory HDL with traditional cardiac risk factors. The relationship between
traditional cardiac risk factors and HDL function in SLE
and RA patients was examined in a bivariate model.
There was significant association with a history of hypertension (P ⬍ 0.0001) and with nonwhite race (P ⫽
0.003). There was no association with a history of
smoking (currently or ever) or with diabetes (Figure 2B).
There was also no association between HDL function
and levels of total cholesterol, HDL cholesterol, LDL
cholesterol, or triglycerides, or age.
We performed logistic regression to examine the
relationship of SLE and RA with proinflammatory
HDL, controlling for traditional cardiac risk factors.
After controlling for these risk factors, the odds ratio
(OR) for proinflammatory HDL was 19.3 (95% confidence interval [95% CI] 4.4–85.3) in SLE patients and
6.18 (95% CI 1.2–32.1) in RA patients compared with
controls (Table 3).
2545
Correlation with ox-LDL. The relationship between HDL function and levels of ox-LDL was examined in SLE, RA, and control subjects. There was a
positive correlation between HDL function and levels of
ox-LDL, using the fluorescence assay method to detect
ox-LDL (r ⫽ 0.37, P ⬍ 0.0001) (Figure 2C). This
correlation was confirmed in a subset of patients in
whom levels of ox-LDL were measured by ELISA (r ⫽
0.47, P ⫽ 0.003) (data not shown). The correlations
between HDL function and ox-LDL were similar in each
of the 3 patient groups: SLE (r ⫽ 0.42, P ⬍ 0.0001),
controls (r ⫽ 0.364, P ⫽ 0.021), and RA (r ⫽ 0.355, P ⫽
0.064).
Correlation with markers of disease activity. The
relationships between HDL function and markers of
disease activity were also examined in SLE and RA
patients. There was a positive correlation between HDL
function and ESR (mm/hour) (r ⫽ 0.32, P ⬍ 0.001)
(Figure 2D). Among SLE patients, however, there were
no other relationships between HDL function and measures of disease activity or damage, such as positivity
(ever) for anti–double-stranded DNA antibody, lupus
anticoagulant (LAC), or anticardiolipin antibody (aCL),
a history of antiphospholipid antibody (aPL) syndrome
(with a history of arterial or venous thrombosis), or
glomerulonephritis. Additionally, there were no significant correlations between HDL function and SELENASLEDAI score (r ⫽ 0.11, P ⫽ 0.07), SDI score, C3 levels,
or disease duration at the time of blood draw.
There were also no associations between HDL
function and most current treatment regimens, including
methotrexate, azathioprine, mycophenolate mofetil, tumor necrosis factor ␣ inhibitors, or cyclophosphamide,
in RA or SLE patients. There was, however, a statistically significant association between HDL function and
Table 3. Logistic regression of the relationship of SLE and RA with
proinflammatory HDL, controlling for traditional cardiac risk factors*
Variable
OR
95% CI
P
SLE (yes, no)
RA (yes, no)
Age (years)
Hypertension (yes, no)
Diabetes (yes, no)
Smoking (ever, never)
Nonwhite (yes, no)
HDL (mg/dl)
LDL (mg/dl)
19.30
6.18
1.01
1.78
2.64
1.28
3.04
1.01
1.00
4.37–85.26
1.19–32.12
0.99–1.04
0.88–3.61
0.52–13.37
0.56–2.93
1.58–5.83
0.99–1.02
0.99–1.00
0.001
0.03
0.26
0.11
0.24
0.55
0.001
0.54
0.34
* Proinflammatory high-density lipoprotein (HDL) was defined as a
score of ⱖ1.0 fluorescence units in the cell-free assay. SLE ⫽ systemic
lupus erythematosus; RA ⫽ rheumatoid arthritis; OR ⫽ odds ratio;
95% CI ⫽ 95% confidence interval; LDL ⫽ low-density lipoprotein.
2546
McMAHON ET AL
Table 4.
Logistic regression of variables associated with proinflammatory HDL in women with SLE*
Variable
OR
95% CI
P
Age (years)
ESR (mm/hour)
Current prednisone dosage ⬎7.5 mg/day (yes, no)
Nonwhite (yes, no)
Cumulative lifetime prednisone dose (gm)
SELENA-SLEDAI score
History of lupus glomerulonephritis (yes, no)
SDI score
Disease duration (years)
C3 level (mm/dl)
Anticardiolipin antibody (yes, no)
Anti-dsDNA antibody (yes, no)
3.73
2.46
2.95
2.82
1.15
0.96
1.31
0.84
0.98
1.00
1.43
0.48
0.875–15.9
1.52–3.97
1.07–8.18
1.19–6.68
0.67–1.98
0.88–1.06
0.45–3.84
0.65–1.09
0.92–1.04
0.98–1.01
0.59–3.45
0.18–1.31
0.08
⬍0.001
0.04
0.02
0.62
0.42
0.62
0.18
0.48
0.76
0.43
0.15
* Proinflammatory high-density lipoprotein (HDL) was defined as a score of ⱖ1.0 fluorescence unit in the
cell-free assay. OR ⫽ odds ratio; 95% CI ⫽ 95% confidence interval; ESR ⫽ erythrocyte sedimentation
rate; SELENA-SLEDAI ⫽ Safety of Estrogens in Lupus Erythematosus; National Assessment version of
the Systemic Lupus Erythematosus Disease Activity Index; SDI ⫽ Systemic Lupus International
Collaborating Clinics/American College of Rheumatology Damage Index; dsDNA ⫽ double-stranded
DNA.
current prednisone dosage; HDL function was more
proinflammatory in those currently receiving prednisone
at a dosage of ⬎7.5 mg/day (mean ⫾ SD 1.03 ⫾ 0.52
versus 0.83 ⫾ 0.49) (P ⫽ 0.002). There was also a
relationship between cumulative prednisone dose and
HDL scores; i.e., SLE and RA patients who had received
a lifetime prednisone dose of ⬍10 gm had lower mean
HDL scores when compared with those who had received 10–20 gm prednisone (0.81 ⫾ 0.36 versus 1.07 ⫾
0.42) (P ⫽ 0.006). Patients who had received a lifetime
prednisone dose of ⬎10 gm had a mean ⫾ SD score of
0.94 ⫾ 0.41, which was not significantly different from
scores in the other 2 groups (data not shown).
Multivariate analysis was performed to determine which variables were most consistently associated
with proinflammatory HDL in SLE patients. In this
analysis, the only significant factors were ESR (OR 2.46,
95% CI 1.52–3.97) (P ⬍ 0.001), current prednisone
dosage ⬎7.5 mg/day (OR 2.95, 95% CI 1.07–8.18) (P ⫽
0.04), and nonwhite race (OR 2.82, 95% CI 1.19–6.68)
(P ⫽ 0.02) (Table 4). When RA patients were included
in the model, ESR, nonwhite race, and current prednisone dosage all remained significant.
Stability over time. We obtained a second blood
draw from a convenience sample of 11 control subjects
and 14 SLE patients at least 6 months after the initial
sampling, to measure the stability of HDL function over
time. The overall mean ⫾ SD HDL score in the group
was 0.82 ⫹ 0.31 at time 2, versus 0.83 ⫾ 0.28 at time 1.
The correlation between the 2 time points was excellent
(r ⫽ 0.88, P ⬍ 0.001). The SELENA-SLEDAI score
changed by ⱖ2 units in 10 of the 14 SLE patients; there
was no correlation between the change in the SELENASLEDAI score from time 1 to time 2 and the change in
HDL function score. In only 1 patient in the SLE group
did an antiinflammatory level of HDL become proinflammatory, and proinflammatory levels of HDL did not
became antiinflammatory in any patient.
DISCUSSION
SLE and RA are associated with an increased risk
of atherosclerosis, with the greater risk in the SLE
group. To our knowledge, the data presented here are
the first to describe proinflammatory HDL in patients
with SLE or RA. In patients with no SLE or RA, these
abnormal HDLs are known to correlate with CAD or
CAD equivalents (11). In this cohort, all 4 patients with
known CAD had proinflammatory HDL, and all had
SLE.
Normal antiinflammatory HDLs play several critical roles in the prevention of atherosclerosis. HDL
removes and transports excess cholesterol from peripheral cells to the liver for removal from the body
(16–19). HDL also plays an antiinflammatory role by
removing ROS from LDL, thus preventing the oxidation
of LDL and the subsequent recruitment of inflammation
mediators to the vessel wall subendothelial space (6,8).
In addition, HDL inhibits the expression of adhesion
molecules (20,21) and cytokines, such as monocyte
chemoattractant protein 1, in endothelial cells (22),
which prevents monocyte movement into the vessel wall.
Proinflammatory HDLs are unable to prevent the oxidation of LDL and the recruitment of monocytes, and
INFLAMMATORY HDL AS A BIOMARKER FOR ATHEROSCLEROSIS IN SLE AND RA
may enhance the inflammatory response (10,23). A flow
chart depicting a conceptual model of the interaction
between HDL and ox-LDL is available at the author’s
Web site (http://rheumatology.med.ucla.edu/).
Links between inflammation, autoimmunity, oxidative damage, and atherosclerosis have been increasingly reported. Recently, levels of proinflammatory oxidized phospholipids have been described as a significant
predictor of coronary artery stenosis in the general
population (24). Our findings, that levels of both proinflammatory HDL and ox-LDL are elevated in SLE and
RA patients, and that proinflammatory HDL correlates
with higher levels of ox-LDL, provide further evidence
of a link between autoimmunity, proinflammatory oxidized phospholipids, and atherosclerosis. Previous studies have also shown that higher levels of ox-LDL in adult
(25,26) and pediatric (27) patients with SLE are associated with arterial disease. Our data suggest that impaired functional capacity of HDL may be responsible,
at least in part, for the increase in LDL oxidation in SLE
patients. Long-term oxidative damage may load both
LDL and HDL with oxidized products, thus overwhelming normal clearance mechanisms and hindering the
ability of HDL to prevent oxidation of LDL.
Although other studies have demonstrated lower
HDL levels in SLE patients than in controls (28), mean
levels of HDL were normal in our cohort, and HDL
functional capacity was independent of serum levels.
HDL particles contain several components that can
prevent LDL oxidation, including Apo A-I (6,18), paraoxonase (29), and platelet-activating factor–acetylhydrolase (PAF-AH) (30). During periods of acute or chronic
inflammation, HDLs that were previously antiinflammatory can become proinflammatory (7,31), with decreased
fractions of paraoxonase, PAF-AH, and Apo A-I (32–
35). Abnormal levels of several HDL components have
previously been described in SLE patients compared
with healthy controls, although none of these studies has
demonstrated abnormal HDL function (36).
Antibodies against HDL (37), Apo A-I (38), and
PAF-AH (11) have also been described in SLE patients.
We tested whether these antibodies play a role in HDL
function by removing Ig that was isolated with HDL.
Proinflammatory effects were strictly confined to the
HDL particles, and were not influenced by Ig (data not
shown). Therefore, although antibodies to ox-LDL and
to HDL may play a protective role in atherosclerosis or
induce lipid abnormalities, they are unlikely to influence
HDL function in this system.
We chose to exclude patients who were taking
statins, because previous studies of proinflammatory
2547
HDL in CAD patients without SLE have demonstrated
that HDL function can be improved, though not normalized, by statin therapy (1,4,39). It is likely that the
number of patients with CAD included in our analysis
was small because most patients with atherosclerosis are
currently treated with statins. Even with this small
sample size, however, there was a significant increase in
proinflammatory HDL in SLE patients with known
CAD over those with no known CAD.
There was a correlation between HDL function
and ESR, which was found in both bivariate and multivariate analyses. This suggests that proinflammatory
HDL could be linked to an acute-phase response. Interestingly, however, despite similar mean ESR levels
between our cohorts with SLE and RA, proinflammatory HDL was seen only half as frequently in patients
with RA. These data are consistent with the lower
frequencies of cardiovascular events in patients with RA
compared with patients with SLE (40).
Proinflammatory HDL function also did not significantly correlate with disease activity, as measured by
the SELENA-SLEDAI score, although the P value was
borderline (⬍0.08) in bivariate analysis. It is possible
that longitudinal data measuring the area under the
curve of the SELENA-SLEDAI scores would correlate
more accurately with HDL function. Alternatively, the
lack of correlation between SELENA-SLEDAI scores
and proinflammatory HDL could mean that our cohort
was not adequately powered, or that the SELENASLEDAI score is a less accurate measure of active
inflammation than the ESR, or that factors other than
inflammation are responsible for the correlations with
proinflammatory HDL. The fact that the HDL scores
were stable over time in patients with changing
SELENA-SLEDAI scores and prednisone dosages also
suggests that elements other than chronic inflammation
may be affecting HDL function. The most likely association would be genetic risk factors, and studies to
correlate certain genes or gene regions with proinflammatory HDL are currently in progress.
There is some speculation that the increased risk
of thrombotic and atherosclerotic events seen in patients
with SLE may be due in part to a cross-reactivity
between antibodies to phospholipids and ox-LDL (41).
Interestingly, there was no association in our cohort
between either proinflammatory HDL or levels of oxLDL and the presence of aCL, LAC, or a history of
thrombosis. Although some lupus cohorts have shown a
significant association between aPL and atherosclerosis
(42,43), several other cohorts have shown no correlation
(40,44). The association between LAC and atheroscle-
2548
McMAHON ET AL
rosis is also unclear; for example, patients who were
positive for LAC in the prospective Hopkins Lupus
Cohort were more likely to develop myocardial infarction than those who were negative (45). There was no
association, however, with aCL, and neither LAC nor
aCL was associated with subclinical atherosclerosis by
carotid ultrasound (45). Thus, aPL alone are unable to
fully account for the development of atherosclerosis
in SLE.
The lack of association between proinflammatory
HDL and aPL seen in our study suggests that the
mechanisms by which proinflammatory HDLs contribute to atherosclerosis differ from those of aPL. For
example, it is possible that aPL contribute to an increase
in cardiovascular events through thrombosis, while
proinflammatory HDL contributes to the formation of
atherosclerotic plaques. Interestingly, 10 of 12 SLE
patients in our cohort with a history of stroke (83%)
were aPL positive, compared with 2 of 4 (50%) with a
history of myocardial infarction. This may also help to
explain the finding that proinflammatory HDL had a
weaker association with a history of stroke than with a
history of CAD in our cohort.
In conclusion, proinflammatory HDL is a novel
biomarker for increased risk of atherosclerosis in patients with SLE and RA. This abnormality is particularly
evident in the SLE patients. Other potential biomarkers
in SLE include elevated levels of ox-LDL (46), antibodies against lipoprotein lipase (26), and anti–ox-LDL
antibodies (47). Our data, along with those of others,
suggest that any one or a combination of these factors
will be highly predictive of risk for atherosclerosis.
Targeting HDL with interventions that restore protective capacity, such as treatment with an Apo A-I mimetic
peptide (48), might be considered for prevention of
atherosclerosis in patients with proinflammatory HDL
and high levels of ox-LDL.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
ACKNOWLEDGMENTS
The authors thank Betty Tsao, PhD, for providing
assistance in the collection and preparation of patient samples,
and Fanny Ebling, PhD, for assistance in the isolation of pure
HDL from patient samples. Additionally, we thank Brian
Skaggs, PhD, for his careful reading of the text and editorial
suggestions.
14.
15.
16.
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