Summarizetion

I need to summarize these articles attached belowWith focusing on the relation between lipid profile tests and ankylosing spondylitis disease

And HLA-B27 gene with changes in lipid profile

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1473
EXTENDED REPORT
Increased disease activity is associated with a
deteriorated lipid profile in patients with ankylosing
spondylitis
V P van Halm, J C van Denderen, M J L Peters, J W R Twisk, M van der Paardt, I E van
der Horst-Bruinsma, R J van de Stadt, M H M T de Koning, B A C Dijkmans,
M T Nurmohamed
……………………………………………………………………………………………………………….
Ann Rheum Dis 2006;65:1473–1477. doi: 10.1136/ard.2005.050443
See end of article for
authors’ affiliations
…………………..
Correspondence to:
M T Nurmohamed,
Department of Internal
Medicine, VU University
Medical Centre, PO Box
7057, 1007 MB
Amsterdam, The
Netherlands; mt.
nurmohamed@vumc.nl
Accepted 23 April 2006
Published Online First
27 April 2006
…………………..
A
Background: Cardiovascular mortality is increased in patients with ankylosing spondylitis. A possible
explanation might be a more prevalent atherogenic lipid profile in patients with ankylosing spondylitis
than in the general population. It has been postulated that inflammation deteriorates the lipid profile,
thereby increasing cardiovascular risk.
Objective: To explore the association between disease activity and lipid profile in patients with ankylosing
spondylitis.
Methods: Disease activity parameters for ankylosing spondylitis and lipid levels (total cholesterol, highdensity lipoprotein cholesterol (HDLc) and triglycerides) were measured in 45 patients with ankylosing
spondylitis for 6 months after starting treatment with leflunomide or placebo. Findings in this treatment
group were compared with those in 10 patients with ankylosing spondylitis treated with etanercept. A
specialised regression model, adjusting for repeated measurements, age and sex, was used to assess the
influence of the disease activity variables on the lipid levels.
Results: Multilevel regression analyses showed significant associations between disease activity
parameters and lipid levels—for instance, an increase of 30 mm at the end of the first hour in erythrocyte
sedimentation rate was associated with a decrease of about 6% in total cholesterol level and a decrease of
about 11% in HDLc levels. Similar significant associations were found between other disease activity
parameters and lipid levels.
Conclusion: Increase in disease activity was associated with decreases in lipid levels. The decrease in HDLc
levels tended to be almost twice as large as the decrease in total cholesterol levels, resulting in a more
atherogenic lipid profile. Hence, effective treatment of disease activity in patients with ankylosing
spondylitis may lower the cardiovascular risk by improving the lipid profile.
nkylosing spondylitis is a chronic inflammatory disease
that affects predominantly men, starts in young
adulthood and results in immobility of the spine and
sacroiliac joints. Although the number of studies investigating mortality in ankylosing spondylitis is limited, many of
these show an increased total mortality in patients compared
with the general population.1–4 Furthermore, this increased
mortality seems to be predominantly caused by cardiovascular disease (CVD), with a twofold increased cardiovascular
standard mortality.5
A possible explanation for this increased cardiovascular
risk is a higher prevalence of conventional cardiovascular risk
factors, such as a more atherogenic lipid profile.6 An
atherogenic lipid profile is characterised by a reduced level
of high-density lipoprotein cholesterol (HDLc) and increased
levels of total cholesterol, low-density lipoprotein cholesterol
and triglycerides. An important prognostic indicator for
(future) CVD is the atherogenic index, which is the ratio of
total cholesterol to HDLc.7–10
Whether or not an atherogenic lipid profile is present in
patients with ankylosing spondylitis is presently unclear.11–13
Moreover, there is growing evidence that inflammation is
associated with deterioration of the lipid profile,14 15 but so far
data for patients with ankylosing spondylitis are lacking.
Hence, we hypothesise that disease activity in inflammatory
diseases, such as ankylosing spondylitis, worsens the lipid
profile, thereby increasing the risk for (future) CVD.
Consequently, lowering the disease activity in patients with
ankylosing spondylitis may have a beneficial effect on the
lipid profile.
Two recent studies,16 17 investigating the safety and efficacy
of treatment with leflunomide and etanercept in patients
with ankylosing spondylitis, gave us the opportunity to assess
the relationship between disease activity and lipid profile in a
total of 55 patients with ankylosing spondylitis, treated with
leflunomide, etanercept or placebo.
METHODS
Patients
All patients included in the study, aged 18–70 years, fulfilled
the 1984 modified New York criteria for ankylosing spondylitis.18 The first population consisted of 45 consecutive
patients with ankylosing spondylitis participating in a
monocentre, randomised, double-blind, placebo-controlled
phase II trial, in which the safety and efficacy of leflunomide
were assessed.17 Thirty patients were treated with the active
compound at a daily dose of 20 mg and 15 patients received
placebo, for a duration of 24 weeks. During this period, other
pharmacological treatments were maintained stable.
Abbreviations: BASDAI, Bath Ankylosing Spondylitis Disease Activity
Index; CRP, C reactive protein; CVD, cardiovascular disease; ESR,
erythrocyte sedimentation rate; HDLc, high-density lipoprotein
cholesterol
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1474
van Halm, van Denderen, Peters, et al
Table 1 Baseline characteristics of participants
Demographic features
Age (years)
Sex (male:female)
Disease activity parameters
BASG
BASFI
BASDAI
ESR (mm/h)
CRP (mg/l)
Lipids
TC (mmol/l)
HDLc (mmol/l)
Atherogenic index
Triglycerides (mmol/l)
Leflunomide or
placebo (n = 45)
Etanercept (n = 10)
42 (11)
32:13
41 (10)
9:1
6.4 (1.8)
5.3 (1.8)
5.5 (1.4)
15 (10–31)*
13 (4–40)*
6.7 (1.6)
4.4 (1.9)
4.9 (1.2)
37 (13–49)*
11 (4.4–29.4)*
4.9 (1.2)
1.1 (0.9–1.4)*
4.3 (3.1–5.5)*
1.2 (0.8–1.8)*
4.4 (0.8)
1.3 (0.3)
3.7 (1.1)
1.1 (0.7–1.6)*
BASDAI, Bath Ankylosing Spondylitis Disease Activity Index; BASFI, Bath
Ankylosing Spondylitis Functional Index; BASG, Bath Ankylosing
Spondylitis Global; CRP, C reactive protein; ESR, erythrocyte
sedimentation rate; HDLc, high-density lipoprotein cholesterol; TC, total
cholesterol.
*Values are mean (SD) or median (interquartile range), as applicable.
The findings of the aforementioned patients with ankylosing spondylitis were compared with those of a second
population consisting of 10 patients with ankylosing spondylitis who were treated for 6 months with etanercept
(25 mg twice weekly).18
Disease activity parameters
The Bath Ankylosing Spondylitis Global score,19 the Bath
Ankylosing Spondylitis Functional Index20 and the Bath
Ankylosing Spondylitis Disease Activity Index (BASDAI),21 all
with a scale from 0 to 10, were determined by an experienced
rheumatologist at baseline and weeks 4, 12 and 24.
Furthermore, the erythrocyte sedimentation rate (ESR) and
C reactive protein (CRP) levels were measured at baseline
and weeks 2, 4, 6, 9, 12, 16, 20 and 24. ESR was determined
with local measurement techniques and expressed in
millimetres per hour (in men ,20 mm/h and in women
,30 mm/h). CRP was measured using a turbidimetric assay
Table 2
supplied by Biomedical
Netherlands) (,10 mg/l).
Diagnostics
30 mm/h ESR
30 mg/l CRP
1-point BASG
1-point BASFI
1-point BASDAI
Lipids
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
TC
HDLc
Atherogenic index
Triglycerides
Statistics
Measuring lipid levels and disease activity parameters in a
particular population at several time points, with variable
time intervals, causes an important statistical problem—that
is, ‘‘repeated measurements within subjects’’. To adjust for
repeated measurements within subjects, a multilevel linear
regression analysis was used. This regression technique
allows for both starting levels and progression over time to
differ between patients. It calculates the regression coefficients of the progression over time of the various lipid levels,
adjusted for repeated measurements within subjects and
variable time intervals.22
Multilevel regression analysis is a longitudinal linear
regression analysis. It combines many cross-sectional linear
regression models into one model of one variable over time.
In this study, we measured the lipid levels over time and
investigated the influence of the various other variables, such
as disease activity parameters, on these lipid levels. Multilevel
regression analysis quantifies this influence, or rather the
association between the two variables, and tests it for
statistical significance. Furthermore, the observed association
between the two variables—that is, lipid levels and disease
activity parameters—was also corrected for age and sex.
One advantage of combining the cross-sectional data of the
various time points into one association is that this increases
Absolute
change (mmol/l)
20.29
20.13
0.29
20.15
20.18
20.06
0.10
20.13
0.01
20.01
0.03
20.05
0.02
20.02
0.08
20.06
20.01
20.00
0.01
20.07
Relative
decrease (%)
p Value
26.0
211.4
6.8
212.6
23.8
25.7
2.4
210.9
0.1
20.7
0.6
24.4
0.4
21.7
2.0
24.5
20.3
20.4
0.2
25.8
0.001*
0.01*
0.09
0.04*
0.001*
0.001*
0.16
0.001*
0.79
0.44
0.47
0.01*
0.62
0.25
0.19
0.01*
0.66
0.71
0.87
0.01*
Atherogenic index, ratio of total cholesterol (TC) to high-density lipoprotein cholesterol (HDLc); BASDAI, Bath
Ankylosing Spondylitis Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional Index; BASG, Bath
Ankylosing Spondylitis Global Score; CRP, C reactive protein; ESR, erythrocyte sedimentation rate.
Influence of disease activity parameters on lipid levels, calculated using multilevel regression analyses correcting for
age, sex and repeated measurements within subjects.
*Indicates significant associations.
www.annrheumdis.com
The
Lipids
Blood samples for lipid measurements were taken after an
overnight fast at baseline and weeks 2, 4, 6, 9, 12, 16, 20 and
24. Serum total cholesterol (,5.0 mmol/l) and triglycerides
(,2.2 mmol/l) were analysed by an enzymatic method using
the appropriate assays supplied by Roche Diagnostics
(Almere, The Netherlands) on a Hitachi 911 analyser
(Roche), according to the instructions of the manufacturer.
Polyethylene glycol-modified enzymes were used for assessing the HDLc levels (in men .0.9 mmol/l and in women
.1.1 mmol/l). The atherogenic index was calculated as the
total cholesterol level divided by the HDLc level.
Influence of disease activity on the lipid levels
Increase in disease
activity parameter
(Apeldoorn,
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Disease activity deteriorates lipid profile in ankylosing spondylitis
the statistical power. Another important advantage of this
method is that it holds into account and corrects for the
possibility that one or a few patients with aberrant values
distort the association between lipid levels and disease
activity—that is, the above-mentioned repeated measurements within subjects. If one or a few patients have, for
example, an extreme level of a certain lipid, it will not distort
the relationship this lipid has with disease activity, because
the multilevel regression analysis will look only at this
relationship and correct for the aberrant starting points.
As the primary goal of this investigation was to study the
relationship between disease activity and lipid levels, it was
not relevant whether the antirheumatic treatment altered
disease activity; hence the data of placebo-treated and
leflunomide-treated patients were put together. The findings
were compared with those of a group of 10 patients with
ankylosing spondylitis treated with etanercept, for whom
similar analyses were conducted.
As the HDLc levels, the atherogenic index and triglyceride
levels were not normally distributed, data were analysed with
the natural logarithms of these values. For clarity, the
regression coefficients for these lipids were retransformed
to geometric means. The multilevel analyses were carried out
with the statistical program MLwiN.23 A p value of (0.05 was
considered significant.
1475
6
A
4
TC (mmol/l)
Atherogenic index
Triglycerides (mmol/l)
HDLc (mmol/l)
2
0
Weeks
B
7
4
BASG
BASFI
BASDAI
RESULTS
Patients
The first group of 45 patients with ankylosing spondylitis (13
women and 32 men), with a mean age of 42 (standard deviation
(SD) 11; range 21–66) years, was followed for 24 weeks. Nine
patients had hypertension; eight of them used antihypertensive
agents. Two patients were treated for hypercholesterolaemia.
Non-steroidal anti-inflammatory drugs were used by 39
patients and prednisone was given to one.
The findings of 10 patients with ankylosing spondylitis
(1 woman and 9 men), with a mean age of 41 (SD 10; range
28–60) years, starting treatment with etanercept, were
compared with those of the first population. One patient
was treated for type 2 diabetes mellitus, one for hypertension
and one for hypercholesterolaemia. Nine patients used nonsteroidal anti-inflammatory drugs.
All pharmacological treatments remained unchanged during the entire observation period. Table 1 shows the baseline
characteristics, including demographic and clinical data.
Lipid levels and disease activity parameters
Figure 1 shows the lipid levels and the disease activity
parameters in patients with ankylosing spondylitis, treated
with placebo or leflunomide (n = 45). We found no significant changes in these variables during the 24-week
observation period. The multilevel regression analyses yielded
several significant associations between lipid level progression over time and disease activity parameters; the height of
the disease activity had a significant influence on the height
of the lipid levels. Higher ESR and CRP levels were
significantly (p,0.001) associated with lower total cholesterol levels, with regression coefficients of 20.01 and 20.01,
respectively. The ln-triglyceride levels also had an inverse
relationship with ESR and CRP, with regression coefficients
of 20.005 and 20.004, respectively. A similar relationship
was observed between ln-HDLc and ESR and CRP levels
(regression coefficients of 20.004 and 20.002, respectively).
Moreover, the disease activity parameters tended to have a
linear relationship with the atherogenic index (p = 0.09).
The relationship between disease activity parameters and
lipid levels, or the influence of disease activity on the lipid
levels, is shown in table 2 as absolute values and percentages.
The influence of ESR and CRP on HDLc levels was almost
1
Weeks
C
40
20
CRP (mg/l)
ESR (mm/h)
0
0
2
4
6
9
12
16
20
24
Weeks
Figure 1 (A–C) Mean lipid levels and disease activity variables during
the 24 weeks of placebo treatment or lefunomide treatment.
Antherogenic index, TC/HDLc; BASDAI, Bath Ankylosing Spondylitis
Disease Activity Index; BASFI, Bath Ankylosing Spondylitis Functional
Index; BASG, Bath Ankylosing Spondylitis Global; CRP, C reactive
protein; ESR, erythrocyte sedimentation rate; HDLc, high-density
lipoprotein cholesterol; TC, total cholesterol.
twice as large as the effect on total cholesterol levels,
resulting in a higher atherogenic index (p,0.001).
The multilevel analyses of patients treated with etanercept
showed that an increased ESR, CRP, Bath Ankylosing
Spondylitis Global score, Bath Ankylosing Spondylitis
Functional Index and BASDAI significantly (p(0.02)
decreased the total cholesterol levels (regression coefficients
of 20.01, 20.02, 20.07, 20.12 and 20.13, respectively).
Higher ESR and CRP levels were associated with lower HDLc
and total cholesterol levels, albeit that the decrease in HDLc
was (again) twice as large as the decrease in total cholesterol
levels, resulting in a higher atherogenic index. Moreover,
increase in other disease activity parameters was also
associated with an increase in the atherogenic index; this
association did not reach significance (p.0.08).
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1476
van Halm, van Denderen, Peters, et al
…………………
DISCUSSION
Our investigation on patients with ankylosing spondylitis
shows that higher disease activity (parameters) is associated
with lower lipid levels and vice versa. This was observed for
total cholesterol, HDLc and triglyceride levels, which were
significantly associated with disease activity parameters as
the ESR, CRP and BASDAI. Moreover, an increase in disease
activity was associated with a decrease in total cholesterol
levels and a more pronounced decrease in HDLc levels,
subsequently resulting in a more atherogenic lipid profile.
The magnitude in which disease activity influences the
lipid levels was limited, which questions its clinical relevance.
However, although the observed influence is small, it should
be noted that ankylosing spondylitis is a chronic inflammatory disease, which means that this small (detrimental)
influence has a potential clinically relevant effect on the
cardiovascular risk for many years. The importance of small
differences in lipid levels over a prolonged period is best
illustrated by several studies in which cardiovascular risk
reduction was found by lowering lipid levels slightly but over
a prolonged period. Firstly, the follow-up of the Framingham
cohort showed that a lower HDLc level of just 0.25 mmol/l
was associated with a 50% higher risk for future vascular
events.24 Secondly, a landmark study on fibrates showed a
22% reduction in the risk for CVD in the group receiving the
active compound versus placebo. The group receiving the
active compound showed only small changes in lipid levels—
that is, 4% decrease in total cholesterol, 6% increase in HDLc
and 31% decrease in triglyceride levels.25 Although these
figures cannot be directly extrapolated to this study, they do
indicate the clinical relevance of small changes in lipid levels.
Growing evidence suggests that inflammation has an
important role in the pathogenesis of CVD, particularly in
atherosclerosis.26 In addition to a postulated direct effect of
inflammation on endothelial cells, mounting evidence
suggests that inflammation can also increase the cardiovascular risk by deterioration of the lipid profile, which is
supported by the findings of a study showing a decrease in
HDLc and apolipoprotein A I levels and an increase in
triglyceride and apolipoprotein B levels during an acutephase response.27 Other investigators found an association
between an increase in lipids as oxidised low-density
lipoprotein cholesterol and proinflammatory cytokines as
CRP, interleukin 6 and tumour necrosis factor a.28 The
findings of the present study confirm these effects of
inflammation on the various lipid concentrations.
High disease activity is characterised by increased cytokine
expression and this could directly lead to altered lipid levels
through effects on the liver or adipose tissue. Moreover, there
may also be an indirect way through various intermediate
factors—for example, metabolic or dietary factors. Patients
with ankylosing spondylitis with high disease activity might
be in a metabolic state comparable to those with rheumatoid
cachexia.29 Decreasing disease activity would improve their
general well-being and physical function, with subsequent
changes in lipid levels. The results of the present study
support this hypothesis, as lower disease activity (parameters) was associated with a more favourable lipid profile.
This investigation is in line with the accumulating evidence
of the intriguing interactions between dyslipidaemia, atherosclerosis and inflammation, showing a worsening of the lipid
profile during increased disease activity. Obviously, more
prospective investigations with cardiovascular end points are
needed to further unravel these relationships.
ACKNOWLEDGEMENTS
We thank Professor Dr YS Smulders and Dr D van Schaardenburg for
their advice and critical reading of the manuscript.
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Authors’ affiliations
V P van Halm, M J L Peters, M van der Paardt, I E van der HorstBruinsma, B A C Dijkmans, M T Nurmohamed, Department of
Rheumatology, VU University Medical Center, Amsterdam, The
Netherlands
V P van Halm, J C van Denderen, M van der Paardt, R J van de Stadt,
M H M T de Koning, B A C Dijkmans, M T Nurmohamed, Department of
Rheumatology, Jan van Breemen Institute Amsterdam, Amsterdam
J W R Twisk, Department of Clinical Epidemiology and Biostatistics, VU
University Medical Centre
Competing interests: None.
Ethical approval: The local ethics committee approved the study.
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Increased disease activity is associated with a
deteriorated lipid profile in patients with
ankylosing spondylitis
V P van Halm, J C van Denderen, M J L Peters, et al.
Ann Rheum Dis 2006 65: 1473-1477 originally published online April 27,
2006
doi: 10.1136/ard.2005.050443
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Originalien
Z Rheumatol
DOI 10.1007/s00393-016-0092-4
© Springer-Verlag Berlin Heidelberg 2016
Redaktion
U. Müller-Ladner, Bad Nauheim
U. Lange, Bad Nauheim
Introduction
Ankylosing spondylitis (AS) is a prototype of spondyloarthropathies characterized by chronic inflammatory arthritis
with spinal and sacroiliac joint involvement. It may cause structural and functional damage to the skeletal system. In
addition, several systems may be affected,
such as the eyes, skin, gastrointestinal
system, heart and vascular structures [1].
There may be anatomical and functional
changes when both cardiac and vascular involvement are present. Cardiac and
vascular involvement include cardiomyopathy (systolic and/or diastolic dysfunction), valvular diseases (aortic valve insufficiency, regurgitation) and conduction disorders. Furthermore, there may
be an increased risk of atherosclerosis
and coronary artery disease [2, 3].
Atherosclerosis is one of the most important causes of mortality and morbidity
in cardiovascular system (CVS) diseases.
Increased carotid intima–media thickness (CIMT) is a convenient, noninvasive marker that can be used to monitor
atherosclerosis in general, as well as
the progression of cardiovascular diseases [4]. Dyslipidaemia is the most
important risk factor for atherosclerosis. Low-density lipoprotein (LDL)
easily infiltrates the endothelium and
renders the tissue sensitive to oxidative
changes, while high-density lipoprotein
(HDL) has anti-atherogenic properties
and provides a protective effect against
cardiovascular disease [5, 6].
Systemic inflammation is thought to
be involved in lipid profile changes in
A. Kucuk · A. Ugur Uslu · A. Icli · E. Cure · S. Arslan · K. Turkmen · A. Toker · M. Kayrak
Division of Rheumatology, Department of Internal Medicine, Meram School of Medicine, Necmettin
Erbakan University, Konya, Turkey
The LDL/HDL ratio and
atherosclerosis in ankylosing
spondylitis
patients with AS [7]. The presence of systemic inflammation causes the release of
free oxygen radicals via immune-mediated mechanisms, which have been suggested to contribute to the pathogenesis of inflammatory disorders. Systemic
inflammation, endothelial inflammation
and increased oxidative stress are important for the development and progression
of atherosclerosis [8]. Ischemic modified
albumin (IMA) is formed as a result of
changes in the metal ion-binding properties of the N-terminal domain of human
serum albumin. IMA has only recently
been introduced but has rapidly become
a marker of both ischemia and oxidative
stress [9].
Our study is the first to investigate
the relationship between CIMT and the
LDL/HDL ratio and also to assess the
IMA levels. The aim of the study is to
investigate CIMT, the LDL/HDL ratio
and the oxidative stress markers IMA
and total oxidant status (TOS), to determine their relationships among patients
with AS and also to evaluate their use in
atherosclerosis assessment.
Materials and methods
Our study was conducted from June 2013
to June 2014 at the Rheumatology Clinic
of the University Faculty of Medicine.
Sixty AS patients diagnosed using the
Modified New York Criteria and 54 ageand gender-matched controls were included [10]. The Ethics Committee for
Clinical Research of our University Faculty of Medicine approved the present
study. Basic laboratory values and de-
mographics, as well as the Bath AS Disease Activity Index (BASDAI) and the
Bath AS Functional Index (BASFI) were
used to evaluate disease activity and were
recorded for both the patients and the
control group. Participants with any immune deficiency, hypertension, diabetes,
acute and/or chronic infection, coronary
artery disease, chronic obstructive pulmonary disease, a history of malignancy
or suspected malignancy and a history of
smoking were excluded from the study.
Biochemical analyses
Coagulated blood samples were collected
from the patients and the controls following a 12-h fast. After suitable centrifugation, the samples were stored at
–80 ºC until testing. Clinical data and
blood samples were collected over a time
period of 6 months. The serum IMA concentrations were analysed by measuring
the complex composed of dithiothreitol
(DTT) and cobalt chloride (CoCl26H2O)
unbound from albumin by the colorimetric method as described by Bar-Or
et al. [11]. Colour development with
DTT was measured spectrophotometrically at 470 nm and compared with
a serum cobalt blank without DTT. The
results were reported in absorbance units
(ABSU). TOS levels were measured using commercially available kits (Rel Assay, Gaziantep,Turkey). Serum TOS levels were expressed as µmol H2O2 equivalent/l [12]. Total cholesterol (TC), triglycerides (TG) and HDL were measured using a Synchron LX20 system (Beckman
Coulter, Brea, CA, USA) and Beckman
Zeitschrift für Rheumatologie
Originalien
Tab. 1
Demonstrative and laboratory data of the patient and the control groups
AS (n = 60)
Controls (n = 54)
P-value
Carotid intima–media
measurements
Age (years)
41.68 ± 10.98
41.80 ± 9.56
0.953
Gender, m/f (%)
45 (75)/15 (25)
32 (59.3)/22 (40.7)
0.174
BMI (kg/m )
27.09 ± 4.68
28.24 ± 3.77
0.148
Hb (g/dL)
14.38 ± 1.80
14.33 ± 1.39
0.852
WBC (×109/L)
8.12 ± 2.53
6.85 ± 1.35
0.001
ESR (mm/h)
13.31 ± 13.51
6.55 ± 4.87
0.001
CRP (mg/dL)
11.53 ± 22.23
3.33 ± 2.41
0.006
Creatinine (mg/dL)
0.75 ± 0.11
0.82 ± 0.48
0.291
ALT (IU/L)
22.41 ± 11.26
23.61 ± 7.84
0.503
TG (mg/dL)
143.85 ± 66.68
133.81 ± 87.81
0.517
TC (mg/dL)
189.91 ± 37.83
183.25 ± 35.77
0.357
HDL (mg/dL)
43.21 ± 10.04
46.79 ± 12.49
0.114
LDL (mg/dL)
117.92 ± 34.39
109.69 ± 33.58
0.219
LDL/HDL ratio
2.85 ± 1.00
2.47 ± 0.90
0.047
TOS (µmol H2O2
equivalent/L)
212.57 ± 149.44
116.70 ± 122.98
0.005
Statistical analysis
The Statistical Package for Social Sciences
(SPSS) for Windows, version 14.0 (SPSS
Inc, Chicago, IL, USA), was used for our
statistical analyses. Continuous variables
were presented as mean ± standard deviation, while categorical variables were
indicated as a number (n) that was expressed as a percent (%). Variables meeting the parametric assumptions were assessed using the t-test and one-way analysis of variance (ANOVA) in independent groups and Tukey’s HSD test in the
intergroup posthoc evaluation, while categorical variables were assessed by chisquare test. Pearson’s correlation analysis
was carried out to test the correlation of
the data. Any p-values that were less than
0.05 were considered statistically significant.
2
IMA ABSU
0.44 ± 0.17
0.32 ± 0.13
< 0.0001 CIMT (mm) 0.99 ± 0.27 0.76 ± 0.25 < 0.0001 AS ankylosing spondylitis, BMI body mass index, TG triglyceride, TC total cholesterol, HDL-C high-density lipoprotein cholesterol, LDL-C low-density lipoprotein cholesterol, Hb haemoglobin, WBC white blood cells, ESR erythrocyte sedimentation rate, CRP C-reactive protein, ALT alanine aminotransferase, LDL/HDL ratio low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio, TOS total oxidative status, IMA ischemic modified albumin, CIMT carotid intima–media thickness, AS ankylosing spondylitis, m male, f female, BMI body mass index, Hb hemoglobin, WBC white blood cells, ESR erythrocyte sedimentation rate, CRP C-reactive protein, ALT alanine aminotransferase, TG triglyceride, TC total cholesterol, HDL high-density lipoprotein, LDL low-density lipoprotein, LDL/HDL ratio low-density lipoprotein/high-density lipoprotein ratio, TOS total oxidative status, IMA ischemic modified albumin, ABSU absorbance units, CIMT carotid intima–media thickness a Mean ± SD Tab. 2 Age, family history, BASFI and BASDAI scores and drug use rates of the patients with AS AS (n = 60) Age at diagnosisa, (years) 33.51 ± 9.16 Family history, n (%) 11 (18.3) BASFIa 2.72 ± 2.16 BASDAIa 4.01 ± 2.14 SLZ, n (%) 24 (40) ETA, n (%) 8 (13.3) ADA, n (%) 8 (13.3) IFX, n (%) 7 (11.7) AS ankylosing spondylitis, BASFI Bath Ankylosing Spondylitis Functional Index, BASDAI Bath Ankylosing Spondylitis Disease Activity Index, SLZ sulfasalazine, ADA adalimumab, IFX infliximab, ETA etanercept a Mean ± SD reagents. HDL levels were assessed using a direct enzymatic method instead of precipitation. LDL levels were calculated using the Friedewald formula when TG levels were lower than 400 mg/dl. If the Zeitschrift für Rheumatologie TG levels were higher than 400 mg/dl, the LDL levels were directly measured. In all cases, imaging was conducted using a high-resolution ultrasound machine (Logiq S6; General Electric, Milwaukee, WI, USA) with a 12-MHz mechanical sector transducer. The intima media thickness of both the right and left arteria carotis communis (CCA) was measured at three points on the far walls in each CCA 2 cm proximal to the CCA bifurcation. The three locations were then averaged to obtain the mean IMT for each side. The average of the twosided measurements was considered as the patient’s overall mean CIMT. Results The general clinical characteristics and demographics of patients with AS are summarized in . Tab. 1. There was nosignificant difference between AS patients and the control group regarding their age, gender and body mass index (BMI; . Tab. 1). The clinical characteristics and the medical treatment of AS patients are displayed in . Tab. 2. IMA was higher in AS patients compared to the control group (0.44 ± 0.17 ABSU and 0.32 ± 0.13 ABSU, respectively; p < 0.0001). A higher TOS was seen in AS patients than it was in the con- Abstract · Zusammenfassung Z Rheumatol DOI 10.1007/s00393-016-0092-4 © Springer-Verlag Berlin Heidelberg 2016 A. Kucuk · A. Ugur Uslu · A. Icli · E. Cure · S. Arslan · K. Turkmen · A. Toker · M. Kayrak The LDL/HDL ratio and atherosclerosis in ankylosing spondylitis Abstract Objectives. In ankylosing spondylitis (AS) patients, cardiac and vascular involvement may manifest as atherosclerosis and coronary artery disease. Systemic inflammation, oxidative stress, increased low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol constitute a significant risk for atherosclerosis. This study investigated the relationship between carotid intima–media thickness (CIMT), LDL/HDL ratio, total oxidant status (TOS; an indicator of oxidative stress) and ischemic modified albumin (IMA; an ischemic marker in AS patients). Patients and methods. Sixty AS patients were diagnosed using the Modified New York Criteria; 54 age- and gender-matched participants were included as controls. CIMT, LDL/HDL ratio, TOS and IMA were measured using the most appropriate methods. Results. IMA was higher in AS patients compared to controls (p < 0.0001). TOS was also increased in AS patients (p = 0.005); as was CIMT (p < 0.0001). The LDL/HDL ratio was also greater in AS patients compared to controls (p = 0.047). A positive correlation was found between CIMT and LDL/HDL ratio among AS patients. Conclusion. Elevated CIMT, IMA and TOS levels suggest an increased risk of atherosclerotic heart disease in AS patients. The LDL/HDL ratio was higher in AS patients compared to controls, and there was a correlation between LDL/HDL ratio and CIMT, albeit statistically weak. Therefore, the LDL/HDL ratio is not a reliable marker to predict atherosclerotic heart disease in AS patients. Keywords Dyslipidemias · Atherosclerosis · Ankylosing spondylitis · Ischemia-modified albumin · Oxidative Stress Das Verhältnis von LDL zu HDL und Atherosklerose bei ankylosierender Spondylitis Zusammenfassung Ziel. Bei Patienten mit Spondylitis ankylosans (AS) kann sich eine kardiale und vaskuläre Beteiligung als Atherosklerose und koronare Herzkrankheit (KHK) manifestieren. Systemische Entzündung, oxidativer Stress, erhöhtes Low-Density-Lipoprotein(LDL)Cholesterin und vermindertes High-DensityLipoprotein(HDL)-Cholesterin stellen ein signifikantes Risiko für Atherosklerose dar. In der vorliegenden Studie wird die Beziehung zwischen Karotis-Intima-Media-Dicke (CIMT), LDL-HDL-Quotient, totalem oxidativem Status (TOS, Indikator für oxidativen Stress) und ischämiemodifiziertem Albumin (IMA, Ischämiemarker bei AS-Patienten) untersucht. trol group (212.57 ± 149.44 µmol H2O2 equivalent/l and 116.70 ± 122.98 µmol H2O2 equivalent/l, respectively; p = 0.005). CIMT was higher in AS patients compared to the control group (0.99 ± 0.27 mm and 0.76 ± 0.25 mm, respectively; p < 0.0001; . Fig. 1). A higher LDL/HDL ratio was seen in AS patients than in the control group (2.85 ± 1.00 and 2.47 ± 0.90, respectively; p = 0.047; . Fig. 2). A positive correlation was observed between CIMT and age, BMI and the LDL/HDL ratio among the patients (. Tab. 3, . Fig. 3). There was no correlation between IMA, TOS and BMI, CIMT, or the LDL/HDL ratio, erythrocyte sedimentation rate, C-reactive protein or the Patienten und Methoden. Anhand der Modified New York Criteria erfolgte die Diagnosestellung bei 60 AS-Patienten; Kontrollen waren 54 in Alter und Geschlecht entsprechende Personen. CIMT, LDL/HDLQuotient, TOS und IMA wurden mit den am besten geeigneten Methoden ermittelt. Ergebnisse. IMA war bei AS-Patienten höher als bei den Kontrollen (p < 0,0001). Auch TOS war bei AS-Patienten erhöht (p = 0,005) sowie CIMT (p < 0,0001). Der LDL/HDL-Quotient war bei AS-Patienten ebenfalls größer als bei den Kontrollen (p = 0,047). Es fand sich eine positive Korrelation zwischen CIMT und LDL/HDL-Quotient bei AS-Patienten. Schlussfolgerung. Erhöhte Werte für CIMT, IMA und TOS sprechen für ein erhöhtes Risiko einer KHK bei AS-Patienten. Der LDL/HDLQuotient war bei AS-Patienten höher als bei den Kontrollen, und es bestand eine – allerdings schwache – Korrelation zwischen dem LDL/HDL-Quotienten und CIMT. Folglich ist der LDL/HDL-Quotient kein reliabler Marker zur Vorhersage einer KHK bei AS-Patienten. leukocyte count. No difference was seen in CIMT, IMA, TOS or the LDL/HDL ratio across the treatment options (between when the patients received antitumour necrosis factor alpha treatment and sulfasalazine). risk for atherosclerosis [14]. CIMT is a reliable and inexpensive marker that has become a predictor of atherosclerosis and can be used to predict cardiovascular events [4]. Our results showed a higher IMA, TOS, CIMT and LDL/HDL ratio in AS patients compared to the healthy control group. We also found a positive correlation between the CIMT and LDL/HDL ratios. However, the correlation between the LDL/HDL ratio and CIMT was statistically weak. Although the underlying reasons for increased atherosclerosis in chronic inflammatory disorders are not fully understood, systemic inflammation exerts important effects on the process. CIMT is used to assess early-stage atherosclerosis Discussion AS is a systemic inflammatory disorder that may involve multiple systems and organs, mainly in the cardiovascular system. Systemic inflammation is involved in the development of atherosclerosis due to its direct effects on the vascular structures and also its influence on the lipid profile [7, 13]. Increased LDL and decreased HDL levels pose an important Schlüsselwörter Dysliplidämie · Atherosklerose · Spondylitis ankylosans · Ischämiemodifiziertes Albumin · Oxidativer Stress Zeitschrift für Rheumatologie Originalien Fig. 1 8 Carotidintima–media thickness (CIMT)amongankylosingspondylitis (AS)andcontrol groups Fig. 2 8 Low-density lipoprotein (LDL)/high-density lipoprotein (HDL) cholesterol ratio among ankylosing spondylitis (AS) and control groups and is known to have a strong association with coronary artery disease [15]. Gupta et al. [16] showed an increased prevalence of CIMT in AS patients compared to their control group. Additionally, Gonzalez-Juanatey et al. [17] demonstrated higher CIMT values in AS patients than Zeitschrift für Rheumatologie in the control group. In another study, Mathieu et al. [18] reported increased CIMT in patients with AS compared to the control group. AS patients are known to be at an increased risk for atherosclerosis, which is influenced by changes in their lipid profile. During systemic inflammation, structural changes in endothelial cells and lipid molecules accelerate the development of atherosclerosis. LDL transfers cholesterol from the liver to the peripheral tissues, and immune system elements are involved in its placement on the vascular wall. In contrast, HDL carries cholesterol to the liver and prevents its adhesion to endothelial cells [19]. Any rise in LDL levels and decreases in HDL levels are important factors in the diagnosis of atherosclerosis. Divecha et al. [20] reported lower HDL levels in AS patients compared to the control group in their study. Mathieu et al. [21] found lower HDL levels in patients with AS than in their control group. Considering the effects of LDL and HDL on atherogenesis, the predictive value of the LDL/HDL ratio regarding the atherosclerotic changes on the vascular wall is even higher. Sathiya et al. [22] observed a greater LDL/HDL ratio in patients with coronary artery disease compared to the control group in their study. Yang et al. [5] demonstrated a relationship between the LDL/HDL ratio and CIMT and carotid plaques. Recently, the ratio of LDL and HDL has become a representative marker of regression and progression in coronary atherosclerosis [14]. The systemic inflammation commonly seen in patients with AS causes increased oxidative stress and impaired endothelial functions. Normally, the antioxidant system and its mechanisms balance the effects of oxidative radicals. Any inconsistency between these systems leads to oxidative stress. TOS is a recently introduced and commonly used marker that can assess these oxidative stress products [23, 24]. In their study, Karakoc et al. [25] found higher TOS levels in AS patients compared to the control group. Endothelial dysfunction is the first sign in the development process of atherosclerosis. The endothelial dysfunction resulting from oxidative stress plays a major role in cardiovascular disease pathogenesis [26, 27]. Changes in IMA levels are thought to be associated with acute and/or chronic hypoxia and oxidative stress. IMA is formed as a result of decreased metal ion- Tab. 3 CIMT Correlation between serum CIMT and age, body mass index and LDL/HDL ratio Age (years) BMI LDL/HDL ratio R 0.737 0.315 0.395 P < 0.0001 0.014 0.002 CIMT carotid intima–media thickness, BMI body mass index, LDL/HDL ratio low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio Fig. 3 8 The relationship between cholesterol ratio and carotid intima-media thickness (CIMT) among ankylosing spondylitis (AS) patients binding capacity due to the effects of free oxygen radicals on albumin [28]. IMA is a considerablysensitive marker, and it can be used as an early indicator of cardiac ischemia [9, 28]. Although there have been no studies on IMA in AS patients, investigations into autoimmune disorders have reported an increased frequency of atherosclerotic heart disease and systemic inflammation, such as rheumatoid arthritis (RA) and systemic sclerosis (SSc). Leitemperguer et al. [29] demonstrated higher IMA values in RA patients compared to the control group, while Montagnana et al. [30] reported increased IMA values in patients with SSc compared to the controls. The cause of 20–40 % of the mortality seen in AS patients is cardiovascular disease, and cardiovascular mortality is increased by approximately two- to threefold in these patients compared to the general population [17, 18]. Atherosclerotic coronary heart diseases account for an important portion of cardiovascular mortality. There are several factors in AS that may lead to atherosclerotic heart disease, including metabolic syndrome, medications, changes in lipid profiles (low HDL levels) and inflammatory processes [31]. The presence of a chronic inflammatory process, increased oxidative stress and the interaction between the two contribute to all stages, including the formation and progression of atherosclerotic plaque and thrombus formation [32, 33]. In our study, the higher CIMT, IMA and TOS values in AS patients compared with the control group may suggest an increased risk of atherosclerotic heart disease in patients with AS. The LDL/HDL ratio was higher in AS patients than it was in the control group, and there was a correlation between the LDL/HDL ratio and CIMT. However, this correlation was statistically weak. In reality, a normal or increased LDL level is not a reliable marker for the risk of atherosclerosis, since LDL is composed of various subtypes; small, dense LDL and oxide subfractions, which are two of these subtypes, are more atherogenic [34, 35]. Thus, LDL subgroup analysis is a more reliable marker used to diagnose atherosclerotic heartdisease. Onthe other hand, development of atherosclerotic heart disease is not all the time prevented by the increment in the HDL [36]. HDL can be easily dysfunctional and even it can gain a pro-inflammatory feature by undergoing oxidation and modification by various oxidant materials [37]. HDL may not show an antioxidant effect as a result of the polymorphism of the paraoxonase enzyme, which is responsible for the antioxidant effect of HDL [38]. Increased HDL leads to a higher amount of lipid transport in the liver; thus, it can show adverse effects [36, 39]. Therefore, even though the LDL/HDL ratio of a patient with normal LDL and high HDL levels is low, there can be high risk of atherosclerotic heart disease. Similarly, the LDL/HDL ratio will be disproportionately high in a patient with high LDL and low atherogenic LDL subtypes. Shah et al. reported that the LDL/HDL ratio was not associated with CIMT, and it did not provide any additional information about atherosclerotic heart disease [40]. In our opinion, the LDL/HDL ratio is not a reliable or good marker to determine the risk of atherosclerotic heart disease. However, it may be beneficial to assess the LDH/HDL ratio together with other markers to determine the risks of atherosclerotic heart disease. Our study has a few limitations. These include the small number of patients, the prospective cross-sectional nature of the study, and the fact that the differences between the patients with mild and severe disease activity as per the BASDAI scoring system were not taken into account. In conclusion, the high levels of CIMT, IMA and TOS that were observed in AS patients in the present study suggest an increased risk of atherosclerotic heart disZeitschrift für Rheumatologie Originalien ease. The LDL/HDL ratio was higher in AS patients than it was the control group, and the correlation between the LDL/HDL ratio and CIMT was statistically weak. The LDL/HDL ratio is therefore not a promising or reliable marker to detect the risk for atherosclerotic heart disease. However, it can be useful to evaluate the LDH/HDL ratio along with other markers to determine the risk for atherosclerotic heart disease. Corresponding address Dr. A. Kucuk Division of Rheumatology, Department of Internal Medicine, Meram School of Medicine, Necmettin Erbakan University Konya, Turkey drademk@yahoo.com Compliance with ethical guidelines Conflict of interest. A. Kucuk, A. Ugur Uslu, A. Icli, E. Cure, S. Arslan, K. Turkmen, A. Toker and M. Kayrak state that there are no conflicts of interest. The Ethics Committee for Clinical Research of Necmettin Erbakan University Faculty of Medicine approved the present study. References 1. Daikh DI, Chen PP (2014) Advances in managing ankylosing spondylitis. F1000PrimeRep 6:78 2. Maghraoui A El (2011) Extra-articular manifestations of ankylosing spondylitis: prevalence, characteristics and therapeutic implications. Eur J Intern Med 22:554–560 3. 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Medicine (Baltimore) 88:358–365 18. Mathieu S, Joly H, Baron Get al (2008) Trend towards increased arterial stiffness or intimamedia thickness in ankylosing spondylitis patients without clinically evident cardiovasculardisease. Rheumatology (Oxford) 47:1203–1207 19. Takata K, Imaizumi S, Kawachi Eet al (2014) Impact of cigarette smoking cessation on high-density lipoprotein functionality. Circ J 78:2955–2962 20. Divecha H, Sattar N, Rumley A et al (2005) Cardiovascular risk parameters in men with ankylosing spondylitis in comparison with non-inflammatory control subjects: relevance of systemic inflammation. Clin Sci (Lond) 109:171–176 21. Mathieu S, Gossec L, Dougados M et al (2014) Cardiovascular profile inankylosing spondylitis: a systematic review and meta-analysis. Arthritis Care Res 63:557–563 22. Sathiya R, Velu VK, Niranjan Get al (2014) A comparative study of serum uric acid levels and lipid ratios incoronary artery disease patients. Int J Biomed Sci 10:124–128 23. Karkucak M, Capkin E, Alver A et al (2010) The effect of anti-TNF agent on oxidation status in patients with ankylosingspondylitis. ClinRheumatol 29:303–307 24. Stanek A, Cieslar G, Romuk E et al (2010) Decrease in antioxidant status of plasma and erythrocytes from patientswith ankylosing spondylitis. Clin Biochem 43:566–570 25. Karakoc M, Altindag O, Keles H et al (2007) Serumoxidative-antioxidative status in patients with ankylosing spondilitis. Rheumatol Int 27:1131–1134 26. Sloten TT van, Henry RM, Dekker JM et al (2014) Endothelial dysfunction plays a key role in increasing cardiovascular risk intype 2 diabetes: the Hoorn study. Hypertension 64:1299–1305 27. JerkicM, LetarteM(2015)Contributionofoxidative stress to endothelialdysfunction in hereditary hemorrhagic telangiectasia. Front Genet 6:34 28. Ali OS, Abdelgawad HM, Mohammed MS et al (2014) Ischemic heart diseases in Egypt: role of xanthine oxidase system and ischemia-modified albumin. Heart Vessels 29:629–637 29. Leitemperguer MR, Tatsch E, Kober H et al (2014) Assessment of ischemia-modified albumin levels in patients with rheumatoidarthritis. Clin Lab 60:1065–1070 30. Montagnana M, Lippi G, Volpe Aet al (2014) Evaluation of cardiac laboratory markers in patients with systemicsclerosis. Clin Biochem 39:913–917 31. Szabo SM, Levy AR, Rao SRet al (2011) Increased risk of cardiovascular and cerebrovascular diseases in individuals withankylosing spondylitis: a population-based study. Arthritis Rheum 63:3294–3304 32. Valente RL, Valente JM, Castro GR de et al (2013) Subclinical atherosclerosis in ankylosing spondylitis: is there a role forinflammation? Rev Bras Reumatol 53:377–381 33. Şerban C, Dragan S (2014) The relationship between inflammatory and oxidative stressbiomarkers, atherosclerosis and rheumatic diseases. Curr Pharm Des 20:585–600 34. Cure MC, Tufekci A, Cure E et al (2013) Lowdensity lipoprotein subfraction, carotid artery intima-media thickness, nitric oxide, and tumor necrosis factor alpha are associated with newly diagnosed ischemic stroke. Ann Indian Acad Neurol 16:498–503 35. Toth PP, Patti AM, Nikolic D et al (2016) Bergamot reduces plasma lipids, atherogenic small dense LDL, and subclinical atherosclerosis in subjects with moderate hypercholesterolemia: a 6 months prospective study. Front Pharmacol 6:299 36. Saleheen D, Scott R, Javad S et al (2015) Association of HDL cholesterol efflux capacity with incident coronary heart disease events: a prospective case-control study. Lancet Diabetes Endocrinol 3:507–513 37. Icli A, Cure E, Cure MC et al (2015) Endocan levels and subclinical atherosclerosis in patients with systemic lupus erythematosus. Angiology. doi:10.1177/0003319715616240 38. Marsillach J, Becker JO, Vaisar T et al (2015) Paraoxonase-3 is depleted from the high-density lipoproteins of autoimmune disease patients with subclinical atherosclerosis. J Proteome Res 14:2046–2054 39. Ronda N, Favari E, Borghi MO et al (2014) Impaired serum cholesterol efflux capacity in rheumatoid arthritis and systemic lupus erythematosus. Ann Rheum Dis 73:609–615 40. Shah AS, Urbina EM, Khoury PR et al (2013) Lipids and lipoprotein ratios: contribution to carotid intima media thickness in adolescents and young adults with type 2 diabetes mellitus. J Clin Lipidol 7:441–445 Impaired Antiatherogenic Functions of High-density Lipoprotein in Patients with Ankylosing Spondylitis Christina Gkolfinopoulou, Efstratios Stratikos, Dimitris Theofilatos, Dimitris Kardassis, Paraskevi V. Voulgari, Alexandros A. Drosos, and Angeliki Chroni ABSTRACT. Objective. Ankylosing spondylitis (AS) is a chronic inflammatory disease associated with increased risk of cardiovascular disease (CVD). High-density lipoprotein (HDL) exerts a series of antiatherogenic properties and protects from CVD. We evaluated whether HDL antiatherogenic properties are impaired in patients with AS. Methods. HDL (apoB-depleted serum) was isolated from 35 patients with AS and 35 age- and sex-matched controls. We measured the antioxidant capacity of HDL, the ability of HDL to induce cholesterol efflux, the activity of HDL-associated enzymes paraoxonase-1 (PON1) and myeloperoxidase (MPO), as well as the ability of HDL to induce Akt kinase activation. Results. HDL from patients with AS had decreased antioxidant capacity and decreased ability to promote cholesterol efflux from macrophages compared to controls. HDL-associated PON1 activity was lower and HDL-associated MPO activity higher in patients with AS compared to controls. Higher MPO activity correlated positively with lower antioxidant capacity of HDL in patients with AS. In addition, HDL from patients with AS had impaired endothelial Akt kinase activating properties that were inversely correlated with the MPO/PON1 ratio and positively correlated with the cholesterol efflux capacity of HDL. Conclusion. HDL from patients with AS displays impaired antiatherogenic properties. Attenuation of HDL properties may constitute a link between AS and CVD. (First Release August 1 2015; J Rheumatol 2015;42:1652–60; doi:10.3899/jrheum.141532) Key Indexing Terms: ANKYLOSING SPONDYLITIS ANTIOXIDANT EFFECT HIGH-DENSITY LIPOPROTEINS CHOLESTEROL EFFLUX CARDIOVASCULAR DISEASE Ankylosing spondylitis (AS) is a chronic inflammatory disease primarily affecting the skeleton, leading to chronic pain in axial and peripheral joints and to functional impairments1. In patients with AS, a higher early mortality of 1.6–1.9 times that of the general population has been reported2,3. Studies of the causes of death among patients with AS identified cardiovascular disease (CVD) as the From the National Center for Scientific Research Demokritos, Agia Paraskevi, Athens; University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas, Heraklion, Crete; Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina, Ioannina, Greece. Funding provided by the General Secretariat of Research and Technology of Greece Grant Synergasia 09SYN-12-897 (to DK and AC); the Ministry of Education of Greece Grant Thalis MIS 377286 (to ES, DK, and AC), and a Hellenic Rheumatology Society grant (to ES, PVV, AAD, and AC). C. Gkolfinopoulou, MSc; E. Stratikos, PhD, National Center for Scientific Research Demokritos; D. Theofilatos, MSc; D. Kardassis, PhD, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology of Hellas; P.V. Voulgari, MD, PhD; A.A. Drosos, MD, FACR, Rheumatology Clinic, Department of Internal Medicine, Medical School, University of Ioannina; A. Chroni, PhD, National Center for Scientific Research Demokritos. Address correspondence to A. Chroni, Institute of Biosciences and Applications, National Center for Scientific Research “Demokritos,” Agia Paraskevi, Athens 15310, Greece. E-mail: achroni@bio.demokritos.gr Accepted for publication May 29, 2015. leading cause2,3. Several studies reported that AS was associated with an increased risk for ischemic heart disease, congestive heart failure, peripheral vascular disease, arterial hypertension (HTN), and cerebrovascular disease4,5. Some causes of heart disease have been proposed to be direct results of AS, such as aortitis and aortic insufficiency with the possible necessity of cardiac surgery, conduction disturbances of the atrioventricular node with a probable subsequent indication for a pacemaker, and myocardial involvement with a possible compromise of left ventricular function6. Analyses of vascular functional and structural variables associated with atheroma development in patients with AS showed increased carotid intima-media thickness (IMT) or flow-mediated dilation, suggesting the presence of increased risk for atherosclerosis7,8. A systematic literature review and metaanalysis proposed that patients with AS appear to be at a higher risk of myocardial infarction2. Overall, these studies indicate that the excess cardiovascular mortality seen in patients with AS could be related to functional or structural arterial abnormalities. Unraveling the mechanisms, as well as identification of new biomarkers of increased risk for atherosclerosis, in patients with AS is important for both prevention and treatment. Numerous clinical and epidemiological studies have Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. 1652 The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532 demonstrated an inverse association between high-density lipoprotein cholesterol (HDL-C) levels and the risk of CVD9,10. HDL exerts a series of antiatherogenic functions, such as the ability to promote cholesterol efflux from artery wall macrophages, antioxidative activity including the ability to protect low-density lipoprotein (LDL) against oxidation, antiinflammatory effects, and protective effects on the vascular endothelium11,12. HDL or its major apolipoprotein, apolipoprotein A-I (apoA-I), can have direct effects on numerous cell types that play roles in cardiovascular and metabolic health including endothelial cells, vascular smooth muscle cells, leukocytes, platelets, adipocytes, skeletal muscle myocytes, and pancreatic β cells13. The various effects of HDL/apoA-I include the modulation of intracellular calcium, oxygen-derived free radical production, the activity of numerous kinases and enzymes and expression levels of various genes13. Previous studies have shown that during the course of chronic inflammatory rheumatic diseases, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), the antiatherogenic properties of HDL are affected. Specifically, patients with RA and SLE were found to have proinflammatory HDL14,15. In addition, the cholesterol efflux capacity of HDL was impaired in RA patients with high disease activity, as well as in SLE patients with the disease under control16,17. Several proteomic studies have shown that a large number of plasma proteins can associate with HDL and this can affect HDL structure and functions18,19. Under conditions of acute-phase reaction, as well as in chronic inflammation, the HDL protein composition is altered, resulting in changes in HDL function20,21. Among the proteins that associate with HDL and play a role in its atheroprotective functions is paraoxonase-1 (PON1), an enzyme linked to the anti-oxidative, antiinflammatory, and endothelial repair–stimulating effects of HDL19,22. Reduced plasma PON1 activity has been proposed to be a risk factor for major adverse cardiac events in humans23. Myeloperoxidase (MPO) is another protein that, like PON1, binds to HDL and is linked to oxidative stress and atherosclerosis24,25. Site-specific oxidation of apoA-I on HDL by MPO has been linked to impairment of cholesterol efflux capacity and acquisition of proinflammatory functions26,27. Further, epidemiological studies have shown an association of plasma MPO with CVD in the general population25. Given the observed impairment of HDL antiatherogenic properties in patients with RA or SLE14,15,16,17, it is tempting to generalize to other chronic inflammatory diseases such as AS. Similarly to AS, both RA and SLE have been associated with excess cardiovascular risk28. In contrast to AS, however, RA and SLE are characterized by the presence of many autoantibodies that may cause systemic inflammation29,30. This pathology is absent in patients with AS29,30. It is therefore possible that HDL atheroprotective properties are not affected in AS and the increased predisposition to CVD for patients with AS is not related to HDL dysfunction. To test this hypothesis, we evaluated the antiatherogenic functions of HDL in patients with AS compared to controls. More specifically, HDL from patients with AS were assessed for their antioxidant properties using a cell-free assay [dichlorofluorescein (DCF) assay]31,32 that has been used to show the impairment of antioxidant properties of HDL in patients with RA and patients with SLE14. Further, we measured the ability of HDL to induce cholesterol efflux from macrophages, considered one of the major antiatherogenic functions of HDL33,34. In addition, the activity of HDL-associated enzymes PON1 and MPO was measured, because previous studies have shown that the impairment of atheroprotective properties of HDL may be associated with changes in PON1 and MPO activity19,26,27,35. Finally, we examined the ability of HDL to induce Akt kinase activation. HDL contributes to cardiovascular protection, particularly in endothelial cells, by inducing intracellular signaling that leads to the activation of diverse kinase cascades13. One of these kinases is Akt, an enzyme that is activated upon phosphorylation. MATERIALS AND METHODS Human subjects. Study subjects were recruited among patients diagnosed with AS and/or followed at the outpatient Rheumatology Clinic of the University Hospital of Ioannina, Greece. Forty patients were evaluated. Patients diagnosed with AS should fulfill the 1984 modified New York criteria36. AS disease activity was measured by the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI)37. A BASDAI score < 4 is considered inactive disease. Exclusion criteria were hereditary dyslipidemia, autoimmune conditions not pertinent to AS, active infection at the time of the assessment, liver or renal disease, malignancy, alcohol abuse, pregnancy, and lactation. Five patients were excluded — 2 for liver steatosis, 2 for renal insufficiency, and 1 for alcohol abuse. All patients were under anti-tumor necrosis factor-α (anti-TNF-α) therapy, while 6 of them were also taking disease-modifying antirheumatic drugs (DMARD) and 1 also received steroids. Controls were recruited who matched patients for age and sex and met the above exclusion criteria. Subjects were defined as having CVD if they had documented history of CVD events, such as myocardial infarction, unstable angina, or cerebrovascular accident. In addition, subjects having a body mass index (BMI) > 30 were classified as obese. The study subjects’
informed consent and approval from the institution’s ethical committee were
obtained.
Serum lipid and apolipoprotein concentrations. Serum was obtained from
blood samples after an overnight fast and stored at –80°C until use. Total
cholesterol, triglycerides, and apoA-I concentrations were determined using
the commercially available reagents Cholesterol LS (Labkit Chemelex SA),
Infinity triglycerides (Thermo), and ApoA1 (Labkit Chemelex SA),
according to the manufacturer’s instructions. HDL-C was determined in
apoB-depleted serum using the Cholesterol LS reagent. LDL cholesterol
(LDL-C) was calculated using the Friedewald equation38.
HDL preparation. The HDL-containing supernatant (apoB-depleted serum)
from subjects’ serum was isolated by the dextran-Mg2+ method as
described39.
DCF assay. The antioxidant capacity of HDL, prepared by the dextran-Mg2+
method, was tested in the presence or absence of oxidized (ox) LDL by the
DCF assay as described31 with some modifications32. DCF-DA (2,7 dichlorofluorescein diacetate; Molecular Probes/Invitrogen) was dissolved in fresh
methanol at 2.0 mg/ml and incubated at room temperature in the dark for 20
min, resulting in the release of DCF. Upon interaction with oxidants, DCF
Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved.
Gkolfinopoulou, et al: Dysfunctional HDL in AS
1653
is oxidized to fluorescent DCF. To compare the HDL antioxidant properties
of affected and unaffected subjects, we normalized for differences in HDL-C
concentration. HDL (final concentration 50 μg cholesterol/ml) in the
presence or absence of oxLDL (final concentration 100 μg cholesterol/ml)
was added into a black 96-well plate in a final volume of 100 μl. The plate
was incubated at 37°C on a rotator for 1 h in the dark. At the end of this
incubation period, 10 μl of DCF solution (0.2 mg/ml) was added to each
well, mixed, and incubated for an additional 2 h at 37°C with rotation in the
dark. Fluorescence was measured with a plate reader (Fluo-Star Galaxy,
BMG LabTech) at an excitation wavelength of 465 nm and an emission
wavelength of 535 nm. All assays were performed in duplicate.
Measurement of PON1 activity. PON1 activity in HDL, prepared by the
dextran-Mg2+ method, was determined using paraoxon as substrate32,40.
Briefly, the assays were performed in a final volume of 250 µl containing 5
µl of HDL, 5.61 mmol/l paraoxon (paraoxon-ethyl, Sigma Aldrich), 2
mmol/l CaCl2, and 100 mmol/l Tris–HCl, pH 8.0. The rate of p-nitrophenol
formed by the hydrolysis of paraoxon was measured by monitoring the
increase in absorbance at 405 nm for 15 min at room temperature in a
microplate spectrophotometer. PON1 activity was expressed as units per liter
of HDL samples. One unit is the activity that catalyzes the formation of 1
µmol p-nitrophenol per minute.
Measurement of MPO activity. The activity of MPO in HDL, prepared by
the dextran-Mg2+ method, was determined as described16 using the
InnoZyme MPO activity assay kit (EMD Chemicals), according to the
manufacturer’s instructions. Briefly, 80 µl of HDL were added to a 96-well
plate with an immobilized polyclonal antibody specific for human MPO.
Activity of captured MPO was measured using a detection reagent
containing tetramethyl benzidine (TMB) and hydrogen peroxide. Following
color development, the reaction was stopped with sulphuric acid and the
absorbance of the oxidized TMB detected at 450 nm.
Cellular cholesterol efflux assay. The cholesterol efflux capacity of HDL
was quantified as described34,41. To begin, J774 mouse macrophages plated
in 48-well plates were labeled with 0.2 ml of labeling medium [0.25 µCi/ml
4(14C)cholesterol in Dulbecco modified Eagle’s medium DMEM (high
glucose) supplemented with 0.2% (w/v) BSA]. Following 24 h of labeling
and washing, cells were equilibrated for 24 h with 0.3 mM
8-(4-chlorophenylthio)- cyclic adenosine monophosphate (cAMP) in 0.2 ml
of DMEM (high glucose) supplemented with 0.2% (w/v) BSA. Subsequently, efflux media containing 2% v/v HDL, prepared by the dextran-Mg2+
method, in DMEM (high glucose) were added for 4 h. At the end of the
incubation, the supernatants were collected and the cells were lysed in 200
μl of lysis buffer (PBS containing 1% (v/v) Triton X-100) for 30 min at room
temperature by gentle shaking. The radioactivity in 50 μl of the supernatant
and 100 μl of cell lysate was determined by liquid scintillation counting. The
percentage of secreted (14C)cholesterol was calculated by dividing the
medium-derived counts by the sum of the total counts present in the culture
medium and the cell lysate. All assays were performed in duplicate. To
correct for plate-to-plate and day-to-day variations, HDL samples from the
same 3 control subjects were included on each plate.
Akt kinase activation assay. The human umbilical vein endothelial
cell–derived line EA.hy926 (300,000 cells/well in 6-well plates) was
cultured in DMEM (10% fetal bovine serum, 2% pen-strep) to 80%
confluence. Cells were starved for 4 h and then treated in the presence or
absence of HDL, prepared by the dextran-Mg2+ method, at a final concentration of 40 µg apoA-I/ml for 20 min42. Western blot assays were performed
using antibodies for phospho-Akt (Ser473) or total Akt (Cell Signaling).
Image quantitation was performed using the ChemiDoc XRS+ Gel Imaging
System (Bio-Rad) and the Image Lab software (Bio-Rad).
Statistical analysis. Statistical analyses were performed using GraphPad
Prism 5 (GraphPad Software) and IBM SPSS Statistics version 22 (IBM
Corp.) software. All data presented are mean ± SD, unless otherwise
indicated. Patient and control groups were compared using Student’s t test
for continuous variables and the chi-square test of association for categorical
variables. Where indicated, p was adjusted for sex, smoking, HTN, obesity,
diabetes mellitus, CVD, and statin use as covariates using general linear
model univariate analysis. Analysis of the distribution of the data by the
D’Agostino and Pearson omnibus K2 normality test showed that, except for
the values obtained from the DCF assay, all other data do not follow a normal
distribution. Therefore, correlations between variables were evaluated using
the Spearman’s correlation coefficient for nonparametric data. P values
< 0.05 were considered significant. RESULTS Study group characteristics. Thirty-five patients with AS were compared to 35 age- and sex-matched controls for alterations in their HDL atheroprotective properties. On average, patients had longstanding disease with low disease activity (BASDAI < 4; Table 1). All patients received treatment with anti-TNF-α drugs (infliximab), 6 also received DMARD (methotrexate), and 1 also received methylprednisolone. There was no statistical difference for C-reactive protein values between patients with AS and controls. The demographic, lifestyle, and clinical characteristics of patients and controls are summarized in Table 1. BMI and percentage of smokers were similar in patients and controls. There were more subjects using statins among patients, but this did not reach statistical significance. Further, there was no significant difference for the presence of CVD, HTN, diabetes, and obesity between patients with AS and controls. Serum total cholesterol, HDL-C, LDL-C, and apoA-I concentrations did not differ statistically between patients with AS and controls. Serum triglyceride levels were lower in patients with AS compared to controls. Similar lipid profiles between patients with AS with inactive disease and controls have been reported43. Disease-related treatment followed by improvement of disease activity has been shown to result in the improvement of lipid profile of patients with AS44,45,46. Antioxidant capacity of HDL. HDL samples were assessed for their antioxidant properties using the DCF assay31,32. In the absence of oxLDL, HDL (used at equal HDL-C concentration) from both patients and controls produced similar fluorescence signal levels, indicating comparable oxidation status. However, in the presence of oxLDL we saw a marked increase in fluorescence signal when using HDL from patients with AS compared to HDL from the control group, indicating that the capacity of HDL to suppress the oxidative potential of oxLDL is reduced in patients with AS (p < 0.0001; Figure 1). Similar results were obtained when the values for patients with AS and controls who were under statin treatment were excluded (p < 0.0001). HDL-mediated cellular cholesterol efflux. Measurement of cholesterol efflux from macrophages using 2% apoB-depleted serum showed that HDL from patients with AS had decreased capacity to promote cholesterol efflux compared to HDL from controls (p < 0.0001; Figure 2A). The cholesterol efflux capacity of HDL in patients with AS remained decreased compared with controls (p < 0.005), even when cholesterol efflux values were normalized for HDL-C levels (Figure 2B). The cholesterol efflux capacity of HDL in patients with AS Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. 1654 The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532 Table 1. Clinical characteristics, lipids, and lipoproteins of patients with AS and controls. Values are mean ± SD unless otherwise indicated. Age, yrs Men, n (%) Disease duration, yrs BMI, kg/m2 CRP, mg/dl BASDAI (0–10) Anti-TNF-α use, n (%) DMARD use, n (%) Steroid use, n (%) Current smokers, n (%) CVD, n (%) HTN, n (%) DM, n (%) Obesity, n (%) Statin use, n (%) Total cholesterol, mg/dl HDL-C, mg/dl LDL-C, mg/dl Triglycerides, mg/dl apoA-I, mg/dl Patients, n = 35 Controls, n = 35 p* 46.1 ± 13.3 30 (86) 19.0 ± 11.1 24.9 ± 4.8 4.9 ± 3.2 3.2 ± 0.7 35 (100) 6 (17) 1 (3) 13 (37) 1 (3) 8 (23) 1 (3) 5 (14) 9 (26) 170.1 ± 38.9 49.9 ± 17.2 105.4 ± 40.2 89.1 ± 45.6 144.1 ± 49.8 45.3 ± 12.6 26 (74) — 24.7 ± 2.3 3.7 ± 3.0 — — — — 12 (34) 1 (3) 3 (9) 0 1 (3) 3(9) 182.5 ± 49.6 49.3 ± 13.7 110.3 ± 48.8 114.8 ± 54.1 165.7 ± 42.0 0.840 0.232 p (adjusted)** 0.806 0.110 0.803 1.000 0.101 0.314 0.088 0.057 0.249 0.855 0.649 0.035 0.054 0.232 0.627 0.491 0.005*** 0.202 *p calculated by Student’s t test for continuous variables and chi-square test of association for categorical variables. **p calculated by general linear model univariate analysis including sex, smoking, HTN, obesity, DM, CVD, and statin use as covariates. *** Adjustment for each covariant independently of the others resulted in p values between 0.016 and 0.047. AS: ankylosing spondylitis; BMI: body mass index; CRP: C-reactive protein; BASDAI: Bath Ankylosing Spondylitis Disease Activity Index; anti-TNF-α: anti-tumor necrosis factor-α; DMARD: disease-modifying antirheumatic drugs; CVD: cardiovascular disease; DM: diabetes mellitus; HTN: hypertension; HDL-C: high-density lipoprotein cholesterol; LDL-C: low-density lipoprotein cholesterol; apoA-I: apolipoprotein A-I. Figure 1. HDL antioxidative capacity in patients with AS and controls. The fluorescence intensity resulting from oxidation of DCFH by test HDL (50 μg cholesterol/ml), prepared by the dextran-Mg2+ method, in the presence or absence of oxLDL (100 μg cholesterol/ml) was measured in a spectrofluorometer as described. ***p < 0.0001. AS: ankylosing spondylitis; C: controls; P: patients; AU: arbitrary units; HDL: high-density lipoprotein; DCF: dichlorofluorescein; oxLDL: oxidized low-density lipoprotein. was also lower compared to controls when the values for patients with AS and controls using statins were excluded (p < 0.0001 and p < 0.05 without or with normalization for HDL-C levels, respectively). PON1 and MPO activities of HDL. Measurement of PON1 and MPO activity using equal HDL volume for each sample showed that the HDL-associated PON1 activity was significantly lower (p < 0.05) and the HDL-associated MPO activity significantly higher (p < 0.05) in patients with AS compared to controls (Figure 3A, C). Normalization of PON1 and MPO activities for HDL-C levels showed that HDL-associated PON1 activity was also significantly lower (p < 0.05) and MPO activity significantly higher (p < 0.05) in patients with AS compared to controls (Figure 3B, D). Similar results were obtained when the values for patients with AS and controls who were under statin treatment were excluded (p < 0.05 for PON1 activity with or without normalization for HDL-C levels; p < 0.05 for MPO activity without normalization for HDL-C levels, and p < 0.005 for MPO activity with normalization for HDL-C levels). When all measurements were adjusted for equal HDL-C levels, the relationship between HDL-associated PON1 or MPO activity and the antioxidant or cholesterol efflux capacity of HDL from patients with AS was examined by Spearman’s rank correlation. While there was no correlation between PON1 activity and antioxidant or cholesterol efflux capacity, neither between MPO activity and cholesterol efflux capacity, the analysis showed that higher MPO activity correlated positively with worse antioxidant function of HDL from patients with AS (r 0.359, p = 0.020). Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. Gkolfinopoulou, et al: Dysfunctional HDL in AS 1655 Figure 2. HDL-mediated cholesterol efflux in patients with AS and controls. The capacity of HDL (2% v/v), prepared by the dextran-Mg2+ method, to promote total cholesterol efflux from J774 mouse macrophages treated with chlorophenylthio-cAMP, was measured as described. All assays were performed in duplicate. A. Values are expressed as % cholesterol efflux of total cell cholesterol. B. Values are expressed as % cholesterol efflux of total cell cholesterol divided by HDL-C concentration in mg/dl. **p < 0.005; ***p < 0.0001. AS: ankylosing spondylitis; C: controls; P: patients; HDL: high-density lipoprotein; HDL-C: HDL cholesterol; cAMP: cyclic adenosine monophosphate. A recent study proposed that the MPO/PON1 ratio could be a useful marker for coronary artery disease (CAD) risk assessment through modulation of HDL properties47. As shown in Figure 3E, HDL-associated MPO/PON1 ratio is significantly higher for patients with AS compared to controls (p < 0.05). Exclusion of values for patients with AS and controls that were using statins resulted in similar results (p < 0.05). Akt kinase-activating properties of HDL. Incubation of endothelial cells with HDL (used at equal HDL-apoA-I concentration) from patients with AS resulted in a reduced ratio of phosphorylated (p) Akt/Akt compared to cells incubated with HDL from controls (Figure 4A), indicating that HDL from patients with AS has reduced capacity (p < 0.005) to promote signaling events through Akt activation. Similar results were obtained after normalization for HDL-C levels (p < 0.001; Figure 4B) and after exclusion of values for patients with AS and controls that were using statins (p < 0.001 for values without normalization for HDL-C levels and p < 0.01 for values after normalization for HDL-C levels). The pAkt/Akt ratio in endothelial cells after treatment with HDL from patients with AS showed an inverse correlation with the HDL-associated MPO/PON1 ratio in patients with AS (r -0.294, p = 0.046). In addition, the pAkt/Akt ratio was positively correlated with the cholesterol efflux capacity of HDL from patients with AS, after normalization for HDL-C levels (r 0.354, p = 0.020). A positive and stronger correlation between the pAkt/Akt ratio and the cholesterol efflux capacity of HDL was also observed for control subjects (r 0.520, p = 0.0007). DISCUSSION Autoimmune rheumatic diseases have been associated with atherosclerosis and increased risk of cardiovascular morbidity and mortality48. The impairment of HDL atheroprotective properties as a mechanism that leads to increased atherosclerotic risk has been studied for the rheumatic diseases RA and SLE14,15,16,17. Extension of these conclusions to AS should, however, be done with caution because of significant differences in the pathology among these diseases. Specifically, RA and SLE are characterized by the presence of many autoantibodies that may cause systemic inflammation, a pathology that is absent from AS29,30. Because systemic inflammation could be the reason behind the impairment of HDL-mediated atheroprotection in RA and SLE, this issue has to be addressed separately in AS. Further, earlier studies reported increased levels of HDL-associated serum amyloid A and reduced plasma PON1 activity in patients with active AS, suggesting impaired HDL antiatherogenic functions45,49,50. In our current study, we show that HDL atheroprotective properties, such as cholesterol efflux ability and antioxidant capacity, are also impaired in patients with AS. Moreover, the atheroprotective properties of HDL in patients with AS are impaired despite the fact that all patients with AS had received disease-related treatment and the disease was clinically under control. This suggests that changes in the functions of HDL can occur even in the absence of marked systemic inflammation and remain persistent even in clinically managed disease. Differences between patients with AS and controls were, in all cases, unaffected by normalization for HDL-C levels, confirming that our results reflect differences in HDL composition and functionality. Specifically, we find that despite normal serum HDL-cholesterol and apoA-I levels, patients with AS have HDL with (1) reduced capacity to promote cholesterol efflux from macrophages, (2) impaired antioxidant properties, (3) decreased PON1 activity, (4) increased Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. 1656 The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532 Figure 3. HDL-associated PON1 and MPO activity in patients with AS and controls. HDL-associated PON1 (A, B) and MPO (C, D) activity was measured using 5 µl and 80 µl of HDL, respectively, prepared by the dextran-Mg2+ method, as described. A. Values are expressed as HDL-associated PON1 activity in u/l. B. Values are expressed as HDL-associated PON1 activity in u/l divided by HDL-C concentration in mg/dl. C. Values are expressed as HDL-associated MPO activity in ng/ml. D. Values are expressed as HDL-associated MPO activity in ng/ml divided by HDL-C concentration in mg/dl. E. HDL-associated MPO/PON1 ratio. *p < 0.05. AS: ankylosing spondylitis; C: controls; P: patients; HDL: high-density lipoprotein; HDL-C: HDL cholesterol; PON1: paraoxonase-1; MPO: myeloperoxidase. MPO activity, and (5) impaired endothelial Akt kinase activating properties. The cholesterol efflux capacity of HDL from macrophages was demonstrated to have a strong inverse association with carotid IMT and the likelihood of angiographic CAD34 and more recently to be inversely associated with the incidence of cardiovascular events in a popula- tion-based cohort33. The proinflammatory/antiinflammatory properties of HDL were shown to distinguish patients with CVD or CVD equivalents from control subjects better than HDL cholesterol51 and were associated with an increased prevalence of carotid plaques and with a higher IMT in patients with SLE52. Therefore, the impairment of cholesterol Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. Gkolfinopoulou, et al: Dysfunctional HDL in AS 1657 Figure 4. Activation of Akt kinase by HDL from patients with AS and controls. A. The property of HDL (40 μg apoA-I/ml), prepared by the dextran-Mg2+ method, to activate the Akt kinase in HUVEC-derived line EA.hy926 was measured as described. Cellular pAkt (Ser473) and total Akt levels were measured by immunoblotting (a representative set of images is shown in the upper panel). Western blots were scanned and quantified by Image Lab software (lower panel). The normalized levels of pAkt against total Akt in the treated cells are shown as fold activation relative to the non-treated cells. B. Values are expressed as levels of pAkt against total Akt in the treated cells with HDL, calculated as fold activation relative to the non-treated cells, divided by the HDL-C amount in µg for each sample. **p < 0.005; ***p < 0.001. C: controls; P: patients; HDL: high-density lipoprotein; HDL-C: HDL cholesterol; AS: ankylosing spondylitis; HUVEC: human umbilical vein endothelial cell; apoA-I: apolipoprotein A-I; pAkt: phosphorylated Akt. efflux and antioxidant capacity of HLD in patients with AS may in part underlie the increased CVD-associated mortality for this patient group. HDL can turn from antiinflammatory to proinflammatory during periods of acute or chronic inflammation because of changes in HDL proteomic and lipidomic composition20,21. Changes in proteomic composition can lead to decreased PON1 activity and increased MPO activity19,27. The latter enzyme also affects HDL cholesterol efflux capacity26,27. Both PON1 and MPO activities have been linked to cardiovascular risk23,25. In our study, we determined lower HDL-associated PON1 activity and higher HDL-associated MPO activity in patients with AS compared to controls. None of the 2 enzyme activities were correlated with the cholesterol efflux capacity of HDL from patients with AS, suggesting that other changes in HDL composition or other factors are responsible for the reduced cholesterol efflux capacity of HDL in these patients. Regardless, the HDL-associated MPO activity was found to associate with the impairment of the antioxidant capacity of HDL from patients with AS, similarly to what has been previously shown for patients with RA15. A previous study suggested that in patients with coronary artery disease the reduced HDL-associated PON1 activity led to the reduction of endothelial Akt phosphorylation at Ser473, inhibition of eNOS-activation, and subsequent loss of the endothelial antiinflammatory and endothelial repair-stimulating effects of HDL22. Another study demonstrated that MPO-oxidized HDL results in lower Akt phosphorylation in endothelial cells, as well as reduced endothelial repair in mice53. HDL from patients with AS showed reduced capacity for endothelial Akt phosphorylation at Ser473 compared to controls. This reduction was not correlated to HDL-associated PON1 or MPO activity when all measurements were adjusted for equal HDL-C levels. However, it was correlated to the HDL-associated MPO/PON1 ratio, indicating a combined effect of changes in HDL composition on HDL-mediated endothelial Akt phosphorylation and subsequent signaling. This is consistent with a recent study that proposed the serum MPO/PON1 ratio as a potential indicator of dysfunctional HDL47. The pAkt/Akt ratio in endothelial cells after treatment with HDL showed a positive correlation with the HDL-mediated cholesterol efflux capacity in controls and a weaker, but statistically significant, correlation in patients with AS. This finding may suggest that in endothelial cells, the efflux of cholesterol can activate signaling pathways and that impairment of HDL cholesterol efflux capacity also affects HDL-mediated signaling. Further studies are needed to elucidate the interactions between HDL-mediated signaling and cholesterol efflux pathways. Personal non-commercial use only. The Journal of Rheumatology Copyright © 2015. All rights reserved. 1658 The Journal of Rheumatology 2015; 42:9; doi:10.3899/jrheum.141532 A limitation of our present study is that we have no pretreatment data for the subjects. Such data would allow the examination of the relationship between inflammation or autoimmunity and HDL atheroprotective functions in patients with AS and whether treatment improves the HDL atheroprotective functions in these patients. Such analyses will be approached in future studies. Overall, this is, to our knowledge, the first study to evaluate antioxidant, cholesterol efflux, and signaling capacity of HDL from patients with AS. The attenuation of HDL properties observed in patients with AS suggests a molecular link between AS and CVD. The fact that the disease was substantially controlled by therapy in our patient group suggests that the impairments in HDL atheroprotective properties observed in patients with AS are not dependent on a persistent highly active inflammatory status. It is possible that specific mediators of chronic immune responses or other genetic factors can influence HDL functions in a persistent manner and do not allow easy recovery to normal levels when the disease is under control. Our results provide novel insight into the increased cardiovascular risk observed in AS2,3,6,7,8, suggesting that AS affects HDL composition and function and this may result in increased atherosclerosis and cardiovascular risk. Therefore, the functional integrity of HDL may be an important, still unexplored longterm prognostic marker to evaluate risk for atherosclerosis and CVD in patients with AS. REFERENCES 1. Braun J, Sieper J. Ankylosing spondylitis. Lancet 2007;369:1379-90. 2. Mathieu S, Gossec L, Dougados M, Soubrier M. Cardiovascular profile in ankylosing spondylitis: a systematic review and meta-analysis. Arthritis Care Res 2011;63:557-63. 3. Papagoras C, Voulgari PV, Drosos AA. Atherosclerosis and cardiovascular disease in the spondyloarthritides, particularly ankylosing spondylitis and psoriatic arthritis. Clin Exp Rheumatol 2013;31:612-20. 4. Han C, Robinson DW Jr., Hackett MV, Paramore LC, Fraeman KH, Bala MV. Cardiovascular disease and risk factors in patients with rheumatoid arthritis, psoriatic arthritis, and ankylosing spondylitis. J Rheumatol 2006;33:2167-72. 5. Szabo SM, Levy AR, Rao SR, Kirbach SE, Lacaille D, Cifaldi M, et al. 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