Increased expression of endothelial lipase in symptomatic and unstable carotid plaques.

Abstract

The aim of this study was to evaluate endothelial lipase (EL) protein expression in advanced human carotid artery plaques (HCAP) with regard to plaque (in)stability and the incidence of symptoms. HCAP were collected from 66 patients undergoing carotid endarterectomy (CEA). The degree of plaque (in)stability was estimated by ultrasound and histology. In HCAP sections, EL expression was determined by immunostaining and the intensity was assessed on a semi-quantitative scale (low: <25%, high: >25% positive cells). Monocytes and macrophages in adjacent HCAP sections were stained with a CD163 specific antibody. High EL staining was more prevalent in histologically unstable plaques (in 33.3% of fibrous plaques, 50% of ulcerated non-complicated plaques and 79.2% of ulcerated complicated plaques; χ(2) test, p = 0.004) and in the symptomatic group (70.8 vs. 42.9% in the asymptomatic group; χ(2) test, p = 0.028). The majority of EL immunostaining was found in those HCAP regions exhibiting a strong CD163 immunostaining. EL in HCAP might be a marker and/or promoter of plaque instability and HCAP-related symptomatology.

Full text

Page 1

ORIGINAL COMMUNICATION
Increased expression of endothelial lipase in symptomatic
and unstable carotid plaques
Matias Trbus ˇic ´ •Monika Riederer•Majda Vuc ˇic ´ •Ivo Lovric ˇevic ´ •
Boz ˇo Krus ˇlin•Martin Gauster•Sonja Mohrenz•Andrea Berghold•
Beate Tiran•Vesna Degoricija•Sas ˇa Frank
Received: 15 April 2011/Revised: 24 June 2011/Accepted: 19 July 2011/Published online: 13 August 2011
? The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract
thelial lipase (EL) protein expression in advanced human
carotid artery plaques (HCAP) with regard to plaque
(in)stability and the incidence of symptoms. HCAP were
collected from 66 patients undergoing carotid endarterec-
tomy (CEA). The degree of plaque (in)stability was esti-
mated by ultrasound and histology. In HCAP sections, EL
expression was determined by immunostaining and the
intensity was assessed on a semi-quantitative scale (low:
\25%, high: [25% positive cells). Monocytes and mac-
rophages in adjacent HCAP sections were stained with a
CD163 specific antibody. High EL staining was more
prevalent in histologically unstable plaques (in 33.3% of
fibrous plaques, 50% of ulcerated non-complicated plaques
The aim of this study was to evaluate endo-
and 79.2% of ulcerated complicated plaques; v2test,
p = 0.004) and in the symptomatic group (70.8 vs. 42.9%
in the asymptomatic group; v2test, p = 0.028). The
majority of EL immunostaining was found in those HCAP
regions exhibiting a strong CD163 immunostaining. EL in
HCAP might be a marker and/or promoter of plaque
instability and HCAP-related symptomatology.
Keywords
High density lipoprotein ? Inflammation ? Atherosclerosis ?
Macrophages
Endothelial lipase ? Carotid artery plaque ?
Abbreviations
HCAPs
EL
HDL
F
Human carotid artery plaques
Endothelial lipase
High density lipoprotein
Fibrous plaque
M. Trbus ˇic ´ and M. Riederer are equally contributed first authors.
V. Degoricija and S. Frank are equally contributed senior authors.
M. Trbus ˇic ´ ? I. Lovric ˇevic ´ ? B. Krus ˇlin ? V. Degoricija
University of Zagreb School of Medicine, Zagreb, Croatia
M. Trbus ˇic ´ ? V. Degoricija (&)
Department of Medicine, Sisters of Mercy University Hospital,
Vinogradska 29, 10000 Zagreb, Croatia
e-mail: vdegoric@mef.hr
M. Riederer ? S. Mohrenz ? S. Frank (&)
Institute of Molecular Biology and Biochemistry,
Center of Molecular Medicine, Medical University
of Graz, Harrachgasse 21/III, 8010 Graz, Austria
e-mail: sasa.frank@medunigraz.at
M. Vuc ˇic ´
University of Zagreb School of Dental Medicine,
Zagreb, Croatia
M. Vuc ˇic ´ ? B. Krus ˇlin
Ljudevit Jurak University Department of Pathology,
Sisters of Mercy University Hospital, Zagreb, Croatia
I. Lovric ˇevic ´
Department of Surgery, Sisters of Mercy University Hospital,
Zagreb, Croatia
M. Gauster ? S. Mohrenz
Institute of Cell Biology, Histology and Embryology,
Center of Molecular Medicine, Medical University of Graz,
Graz, Austria
A. Berghold
Institute for Medical Informatics, Statistics and Documentation,
Medical University of Graz, Graz, Austria
B. Tiran
Clinical Institute for Medical and Chemical Laboratory
Diagnostics, Medical University of Graz, Graz, Austria
123
J Neurol (2012) 259:448–456
DOI 10.1007/s00415-011-6198-3

Page 2

UNC
UC
CEA
CDFI
CTA
CRP
Ulcerated non-complicated
Ulcerated complicated
Carotid endarterectomy
Color Doppler Flow Imaging
Computed tomography angiography
C-reactive protein
Introduction
Atherosclerosis is a chronic fibro-proliferative and immuno-
inflammatory disease affecting large and medium size
arteries. It is characterized by endothelial dysfunction,
local oxidation of circulating lipoproteins [particularly low
density lipoprotein (LDL)] and their accumulation in the
vascular wall [1, 2], and the activation of proinflammatory
cytokines [3, 4]. Inflammation plays a crucial role in plaque
destabilization, as it converts stable atherosclerotic pla-
ques, characterized by a fibrous cap covering the necrotic
core, into unstable ones, characterized by a thinned fibrous
cap, intra-plaque hemorrhage, surface ulceration, rupture
and thrombosis [5].
Endothelial lipase (EL) is a member of the triglyceride
lipase gene family, expressed by vascular endothelial cells
(ECs), smooth muscle cells (SMCs) and macrophages [6, 7].
It primarily exhibits phospholipase-A1 activity, with high
affinity for high density lipoprotein (HDL)-phosphatidyl-
choline (HDL-PC), resulting in the generation of bioactive
molecules such as lysophosphatidylcholines (lyso-PC) and
free fatty acids (FFA) [8]. Acting on HDL, EL not only
promotes HDL remodeling but also lowering of HDL plasma
levels, an independent risk factor for atherosclerosis [9]. EL
plasma concentrations are strongly associated with elevated
plasma interleukin-6 (IL-6), tumor necrosis factor (TNF)-a
and C-reactive protein (CRP) levels; a profile typical of
metabolic syndrome [10, 11]. EL was also shown to promote
the expression of inflammatory cytokines, exemplified by
attenuation of inflammatory cytokine expression in EL-defi-
cient macrophages [12] as well as by increased production of
IL-8 in human endothelial cells over-expressing EL [13].
Taking into account the role of inflammation in plaque
destabilization, together with the reported up-regulation of
EL under inflammatory conditions, we assumed a positive
relationship between the expression level of EL and the
degree of plaque instability.
Patients and methods
Patients
Patients were recruited from the Department of Vascular
Surgery, Sisters of Mercy University Hospital, Zagreb,
Croatia, between 1 November 2007 and 1 March 2009.
Written informed consent from each patient was obtained
prior to the enrollment in the study, which was performed
according to Good Clinical Practice and Helsinki Decla-
ration principles. The study was approved by the local
ethics committee, in accordance with institutional guide-
lines of the Sisters of Mercy University Hospital, Zagreb.
Inclusion and exclusion criteria
Overall, 88 patients with significant (70–99%) carotid
artery stenosis (confirmed by carotid Color Doppler Flow
Imaging (CDFI) done by an experienced physician in the
University Hospital) were screened for inclusion and
exclusion criteria. In cases where CDFI imaging was lim-
ited by features such as calcified carotid lesions, tortuous or
kinked carotid arteries or patient body constitution, contrast
enhanced magnetic resonance angiography (CEMRA) or
computed tomography angiography (CTA) was performed.
The clinical indication for carotid endarterectomy (CEA)
was met after examination by the neurologist and vascular
surgeon, based on clinical presentation and CDFI (or
CEMRA/CTA) results, following guidelines for treatment
of extra-cranial carotid artery disease [14, 15]. Exclusion
criteria were met in case of acute or chronic infection or
inflammatoryconditions (other
severe renal failure (serum creatinine C200 mmol/L),
severe hepatic cirrhosis (Child-Pugh Class B or C), neo-
plasms, recent trauma or surgery, history of ipsilateral CEA
or radiotherapy and anticoagulant therapy. Patients with a
possible cardiac source of embolism detected through the
patients’ history, electrocardiogram or echocardiography
(left ventricular aneurysm, previous large anterior cardiac
infarction, dilatative cardiomyopathy, aneurysm of inter-
atrial septum with patent foramen ovale) were excluded
from the study.
than atherosclerosis),
Definition of symptomatic carotid disease
The carotid stenosis was considered symptomatic if a
patient had experienced a focal neurological symptom
(ipsilateral stroke, transient ischemic attack or monocular
blindness), sudden in onset and referable to the respective
carotid artery within 4 months of CEA.
Patient characteristics, history and medication
Medical history was recorded from all patients, and the
presence of vascular risk factors, features of metabolic
syndrome,previous antihypertensive,statin,andantiplatelet
treatments were noted. Clinical history was assessed for
diabetes mellitus, smoking, hypertension, hyperlipidemia,
prior acute myocardial infarction, symptoms of peripheral
J Neurol (2012) 259:448–456 449
123

Page 3

vascular disease and family history of CHD or IVC in first-
degree relatives under the age of 55 years. The body mass
index (BMI) was calculated according toQuetelet’s formula
as the ratio of body weight to body height squared (kg/m2).
Waist circumference was measured with a flexible tape,
placed on a horizontal plane at the level of the iliac crest.
Metabolic syndrome was defined according to the criteria of
the International Diabetes Federation issued in 2005 (http://
www.idf.org/): increased waist circumference (for Cauca-
sian European men C94 cm, for women C80 cm) plus any
two of the following: triglycerides[1.7 mmol/L (or treat-
ment for elevated triglycerides), HDL cholesterol \1.03
mmol/L in men or\1.29 mmol/L in women (or treatment
for low HDL), systolic blood pressure [130 mmHg,
diastolic blood pressure [85 mmHg (or treatment for
hypertension), and fasting plasma glucose[5.6 mmol/L (or
previously diagnosed type 2 diabetes mellitus) .
Blood pressure was measured with mercurial sphyg-
momanometer (Riester, Jungingen, Germany) and calcu-
lated as the average of three measurements taken under
standardized conditions in a supine position. Hypertension
was diagnosed according to World Health Organization
(WHO) criteria (blood pressure C140/90 mmHg or current
anti-hypertensive treatment). Diabetes was diagnosed in
patients with dietary treatment, anti-diabetics or current
fasting plasma glucose levels higher than 7.0 mmol/L.
Hyperlipoproteinaemia was defined if the low density
lipoprotein (LDL) cholesterol level was[3.5 mmol/L, or if
a patient was taking a lipid-lowering drug. According to
cigarette smoking, patients were classified as current
smokers (smoking more than five cigarettes within the past
3 months), former smokers ([3 months and\10 years) or
non-smokers.
Methods
Color Doppler Flow Imaging (CDFI)
CDFI of both carotid arteries was performed in all subjects
on admission using commercially available equipment
(Aloka 5,500 and Alfa 10 Premium with 10-MHz linear
array transducer). The grade of plaque stenosis was
assessed according to defined criteria [16]. Plaques were
divided depending on the presence of the echolucency and
calcification into: (1) fibrous plaques (without echolucent
material and calcification), (2) soft plaques (predominantly
echolucent material), (3) calcified plaques, (4) mixed pla-
ques (both calcification and echolucent material present),
and (5) non-defined plaques (fibrous plaques with small
degree of echolucency and calcification). In this regard,
echolucency is considered an important factor associated
with symptomatic carotid
vulnerability.
disease [17] andplaque
Tissue sampling, preservation of carotid plaque specimens
and histology
Carotid specimens were excised by the vascular surgeon
(complete tubular specimen without damage to the luminal
surface) and collected during surgery. They were imme-
diately rinsed in 0.9% saline, fixed for 24 h in 5 mL buf-
fered formalin, followed by several phosphate buffered
saline (PBS) washes and storage in PBS for 2 weeks before
embedding in paraffin. Plaque morphology was assessed by
standard histological examination of hematoxylin and
eosin-stained (HE) sections (5 lm) and screened for fea-
tures indicative of plaque vulnerability. Taking into
account the lack of new, prospectively validated criteria for
plaque vulnerability, the Stary classification [5] was
applied and plaques were defined as: (1) fibrous (F) (cov-
ered by a smooth luminal surface indicative of an intact
fibrous cap), (2) ulcerated non-complicated (UNC) (thin
fissured or ruptured cap), and (3) ulcerated complicated
(UC) (thin fissured or ruptured cap with intra-plaque
hemorrhage and/or thrombus).
Immunohistochemistry
Formalin fixed and paraffin embedded human carotid sec-
tions (5 lm) were mounted on Superfrost Plus slides
(Menzel, Braunschweig, Germany) and dried at 40?C
overnight. Slides were dewaxed twice in xylene for 10 min
and rehydrated through a graded series of alcohol. Antigen
retrieval was performed by sequentially incubating the
slides in antigen retrieval buffer pH 9 (Eubio, Vienna
Austria) for 2 min at 720 W and 3 9 5 min at 160 W in
the microwave. Immunohistochemistry was performed
using the Ultravision LP detection system (Thermo Sci-
entific, Fremont, USA) according to the manufacturer’s
instructions. In brief, endogenous peroxidase was blocked
by incubation with Hydrogen Peroxide Block (Thermo
Scientific) for 10 min. Thereafter, slides were washed with
PBS and unspecific background was blocked with Ultra V
Block (Thermo scientific) for 8 min. Rabbit clonal anti-
human CD163 (DB Biotech, clone K20-T, diluted 1:100)
and rabbit anti-EL serum (1:1,500) [18] were diluted in
antibody diluent (DAKO) and incubated on slides for 1 h at
RT. After PBS washing steps, detection was achieved by
incubation with the UltraVision HRP-labelled polymer
system for 15 min and 3-amino-9-ethylcarbacole (AEC,
Dako, Denmark), according to the manufacturer’s instruc-
tions. For negative controls, slides were incubated with
rabbit IgG Ab-1 (2 lg/ml, Neomarkers) instead of the
primary antibodies. Nuclei were stained with hemalaun and
slides were mounted with Kaiser’s glycerol gelatine. Ima-
ges of sections were taken with an Axiophot microscope
equipped with an AxioCam HRc digital camera (Zeiss,
450 J Neurol (2012) 259:448–456
123

Page 4

Oberkochen, Germany). Importantly, all samples were
processed simultaneously using the same batch of reagents.
The expression of EL was evaluated on a semi-quantitative
scale by three independent, experienced investigators
blinded to the clinical and histology data. The scoring was
arbitrary, as follows: (1) low staining (A) (\25% positive
cells), and (2) high staining (B) ([25% positive cells).
Assays
Blood samples for laboratory assays were obtained before
surgery at approximately 8 am, following overnight fast-
ing. Blood sampled in ethylenediaminetetraacetic acid
(EDTA)-K3 containing tubes was used for hematological
assays (Coulter-Counter S plus junior, Coulter Electornics
Limited, Luton, UK). Coagulation parameters were asses-
sed from blood samples in 3.8% sodium citrate. After
separation of plasma by centrifugation (3000g for 10 min),
the Quicktest was performed on a Behring coagulation
timer (Dade Behring, Marburg, Germany). Serum creati-
nine, bilirubin, aspartate transaminase, alanine transami-
nase, blood glucose level, total plasma cholesterol,
triglyceride, LDL and HDL were measured on automated,
multi-channel selective analyzers Olympus AU2700 and
Olympus Fractoscan junior (Olympus Diagnostica GmbH,
Hamburg, Germany). High sensitivity (hs) CRP concen-
trations were measured on automated, multichannel selec-
tive analyzer Modular (Roche Diagnostics, Mannheim,
Germany). IL-6 concentrations were measured using a
specific chemiluminescent ELISA (QuantiGlo; R&D Sys-
tems, Wiesbaden-Nordenstadt, Germany) according to the
manufacturers’ instructions.
Statistical analysis
Data were summarized using mean and standard deviation
or median, minimum, maximum as appropriate for con-
tinuous variables and counts as well as percentages for
categorical variables. Data distribution was analyzed by
Kolmogorov–Smirnov test. According to these results,
appropriate parametric or non-parametric tests were used
in further analyses. Unpaired Student’s t-test or Mann–
Whitney U test was performed to assess differences
among quantitative variables between symptomatology and
immunohistochemistry groups. Differences between ordi-
nally scaled histological groups were analyzed with Krus-
kal–Wallis test, whereas Chi square test was applied to
analyze differences in qualitative and categorical variables.
The association between quantitative data was evaluated
using Spearman-rank correlation analysis. Data were
analyzed using PASW 17.02 (IBM, Chicago, IL, USA)
software and all p values below 0.05 were considered
significant.
Results
Sample size and clinical characteristics
Out of 88 patients assessed for eligibility, 22 patients were
excluded (see Fig. 1). The final study population included
66 patients (43 males and 23 females, mean age 67.5 years)
stratified into two groups: the asymptomatic group (42 pts)
and the symptomatic group (24 pts). The patients’ clinical
characteristics and medications are shown in Table 1. The
two groups did not differ significantly according to age,
gender, individual risk factors, medication, or coexistence
of other vascular diseases (peripheral vascular disease, past
myocardial infarction, angina pectoris). In the symptomatic
group, however, a higher incidence of patients with
increased waist circumference was found (62.5 vs. 38%; v2
test, p = 0.049). The BMI was also higher in the symp-
tomatic group (28.2 ± 3.0 vs. 26.8 ± 3.0) but the differ-
ence did not reach statistical significance (p = 0.085).
Overall, there was no significant difference in the number
of metabolic risk factors. Basic biochemical and hemato-
logical parameters as well as inflammatory parameters
(WBC, hs-CRP, erythrocyte sedimentation rate, fibrinogen
and IL-6) did not differ between the groups (Table 2).
Fig. 1 Flow chart of the study
J Neurol (2012) 259:448–456451
123

Page 5

Ultrasound and histological characteristics of plaques
The grade of carotid artery stenosis detected by CDFI (or
CEMRA/CTA) was considerably higher in the symptom-
atic group (v2test, p = 0.027) (Table 3). Additionally,
plaques with morphologic characteristics suggestive of
plaque vulnerability (echolucent zones with or without
ulcerated morphology) seen on CDFI were more frequent
in the symptomatic group (v2test, p = 0.039). Finally, the
majority of asymptomatic plaques (50%) were F plaques,
whereas the majority of symptomatic plaques (50%) were
UC plaques (not statistically significant) (Table 3).
EL immunostaining in plaques
In F plaques (Fig 2a–c), the necrotic core was covered with
a fibrotic cap with preserved endothelium and only few
inflammatory cells (Fig. 2a). In UC plaques (Fig. 2d–e), in
contrast, the necrotic core was covered with inflammatory
infiltrates, composed of lymphocytes, macrophages, sid-
erophages and importantly of erythrocytes, indicating intra-
plaque hemorrhage (Fig 2d). In F plaques, a weak EL
immunostaining (type A) was detected in the endothelial
layer and in the fibrous cap (Fig. 2b). The fibrous cap also
exhibited moderate CD163 immunostaining, indicative of
the presence of monocytes and macrophages (Fig. 2c).
Compared with F plaques, EL staining was more pro-
nounced (type B) in UC plaques (Fig. 2e). Here, EL
staining localized primarily to cells of the inflammatory
infiltrate (Fig. 2e), which showed a strong staining for
CD163 as well (Fig. 2f).
Importantly, high intensity EL immunostaining (type B)
was most frequently observed in UC plaques (79%; 19 of
24 pts), followed by UNC and F with 50% (6 of 12 pts) and
33% (10 of 30 pts), respectively (Fig. 3) (v2
p = 0.004). Furthermore, EL immunostaining type B was
test,
Table 1 Clinical characteristics
and patient medications at the
enrollment into the study
BMI body mass index, ACE
angiotensin-converting enzyme
aFamiliar history of
cardiovascular disease or stoke
in first relatives younger than
55 years
?Fisher exact test, p = 0.049
Clinical characteristic and medicationsAsymptomatic (n = 42)
(mean ± SD)
Symptomatic (n = 24)
(mean ± SD)
Age (years; median, range)
BMI (kg/m2; mean ± SD)
69 (43–83) 65.5 (50–84)
26.8 ± 3.028.2 ± 3.0
Waist circumference (cm; mean ± SD)91.6 ± 7.5194.1 ± 8.5
No. of patients (%)
Gender
Men28 (66.6)15 (62.5)
Women
Increased waist circumference?
14 (33.3)9 (37.5)
16 (38.1)15 (62.5)
Cardiovascular risk factors
Arterial hypertension36 (85.7)22 (91.6)
Hyperlipoproteinaemia31 (73.8)21 (87.5)
Diabetes mellitus or glucose intolerance20 (47.6)15 (62.5)
Tobacco (current or recent)
Family historya
16 (38.0) 14 (58.3)
9 (21.4)4 (16.7)
Peripheral vascular disease21 (50.0) 9 (37.5)
Coronary artery disease12 (28.5)6 (25.0)
Medications
Statins29 (69.0)16 (66.6)
ACE inhibitors21 (50.0)12 (50.0)
Angiotensin receptor blockers7 (16.6)3 (12.5)
Calcium channel blockers19 (45.2) 8 (33.3)
Acetyl salicylic acid 25 (59.2) 19 (79.1)
No. of metabolic elements present
03 (7.1)1 (4.2)
1 4 (9.5)2 (8.3)
2 15 (35.7)5 (20.8)
38 (19.0)4 (16.7)
49 (21.4)11 (45.8)
5 3 (7.1)1 (4.2)
452J Neurol (2012) 259:448–456
123