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VASCULAR SURGERY
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ORIGINAL RESEARCH
Relationship between white blood cell count and
circulating anti-endothelial cell antibodies detected in
patients with peripheral arterial disease
Cesar Varela MD, Joaquin de Haro MD, Leticia Esparza MD, Silvia Bleda MD,
Ignacio Lopez de Maturana MD & Francisco Acin Md PhD
Angiology and Vascular Surgery Department. Hospital Universitario de Getafe. Crta.
Toledo KM 12.5, 28905, Getafe (Madrid), Spain.
Date received: 5/5/12, Date accepted: 12/7/12
Key words: Anti-endothelial cell antibodies; peripheral arterial disease; monocyte; eosinophil; inflammation
DOI: 10.5083/ejcm.20424884.83
ABSTRACT
CORRESPONDENCE
Background and objectives: Circulating anti-endothelial cell antibodies (AECAs) are elevated in
peripheral arterial disease (PAD) patients. In this context, these autoantibodies have been associated
with the endothelial dysfunction and the pro-inflammatory status that surrounds atherosclerosis.
On the other hand, white blood cell (WBC) count and inflammatory markers have been consistently
associated with cardiovascular events and PAD. Our aim is to investigate the relationship between
circulating AECAs and WBC count in patients with PAD.
Cesar Varela MD
Av. de Aragón 36, 3ºC, 28903,
Getafe (Madrid), Spain
Tel: 034-626-98-50-93
Fax: 034-91-764-15-90
E-mail: [email protected]
Methods: An observational translational study was conducted including 34 male patients with
intermittent claudication and no previous autoimmune disease. WBC count was measured in all
patients. Highly sensitive C-reactive protein levels (hsCRP) were investigated as a surrogate of
inflammation. Circulating AECAs titer was detected using indirect immunofluorescence.
Results: We found higher hsCRP levels in patients with circulating AECAs (7.60 [4.25-10.20] vs. 4.90
[3.20-7.00] mg/L p=0.02). We also observed a higher monocyte count (0.72 [0.60-1.01] vs. 0.55
[0.45-0.79] X103/µL p=0.03) and eosinophil count (0.28 [0.22-0.36] vs. 0.25 [0.07-0.29] X103/µL
p=0.05) in patients with these autoantibodies
CONFLICT OF INTEREST
None
Conclusions: Circulating AECAs of PAD patients could be associated with the systemic inflammatory
status that surrounds the disease and with high monocyte and eosinophil blood cell count.
INTRODUCTION
Atherosclerosis is a chronic inflammatory vascular disease based on the immune system activity. Therefore, complex interactions between genetic and environmental factors of the immune
system and the vascular wall could act as modulators of the pathogenesis and development of
peripheral arterial disease (PAD).
Indeed, white blood cell count (WBC) and inflammatory markers elevation have been consistently associated with cardiovascular events
and PAD (1-5). Moreover, there are several data
pointing to a possible autoimmune origin for
atherosclerosis (6-11).
In this context, circulating anti-endothelial cell
antibodies (AECAs) are a heterogeneous group
of circulating antibodies targeting endothelial
cells, detected in different vasculitic, inflammatory or autoimmune situations, whose common
denominator is endothelial damage (12).
AECAs have various physiopathological effects
of scarcely known mechanisms, such as induction to apoptosis (13,14), cytotoxicity (15), increase
in leukocyte adhesion and cytokine secretion
(16,17), activation of coagulation and thrombosis.
In a recent study it has been found an association between these autoantibodies and PAD in
patients with no previous autoimmune disease
(18). Our aim is to investigate the relationship between circulating AECAs and WBC count in patients with PAD in order to provide new insights
in the field of the etiological pathophysiology of
atherosclerosis.
ISSN 2042-4884
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METHODS
An observational translational study was conducted including
male patients with intermittent claudication due to PAD after haemodynamic confirmation of the disease by Doppler and treadmill
exercise testing. None of the patients included had been previously
revascularised or presented tissue lesions of the lower limbs. All patients signed an informed consent form, according to the principles
of the Helsinki Declaration and the study protocol was approved by
our Hospital Ethics Committee.
Figure 1: Circulating anti-endothelial cell antibodies
(AECAs) detection. Positive reaction is defined as granular
fluorescence in cytoplasm of cultivated human endothelial
cells from umbilical vein.
All those patients with documented diagnosis of atopic, autoimmune or rheumatologic disorders, transplanted or immunodepressed, and those treated with immunosupressors or systemic
and/or inhaled corticosteroids were excluded. We also excluded
all those patients with an acute inflammatory status. Remaining
subjects underwent a serological screening for autoimmune, rheumatologic and neoplasic disease markers. Antibodies ANA, AntiDNA, Anti-LKM, Anti-mitochondria and Anti-smooth muscle were
analysed by means of indirect immunofluorescence and antibodies
Anti-SM, Anti-Jo, Anti-LA, Anti-Scl 70, Anti basement membrane,
Anti-RO and Anti-RNP were measured using the enzyme-linked
Immunosorbent assay (ELISA) test. All patients seropositive to any
marker were excluded from the analysis.
During two months, 248 patients were screened at our outpatients clinic, but only 34 met the inclusion criteria and were finally
recruited for the research. The basal ankle-brachial-index higher
value and the absolute claudication distance of the included subjects were 0.60 [0.57-0.70] and 175 [126-236] metres, respectively.
Cardiovascular risk factors and medical treatment of the patients
were recorded.
Basic laboratory, WBC and highly sensitive C-reactive
protein measurements
Patients who met the inclusion criteria were called to attend our
outpatient clinics after fasting for 12 hours. We obtained a general
sample for basic laboratory determinations (glycaemia, electrolytes
and renal function), lipid profile and WBC count. Using the same
puncture, we also obtained an independent blood sample for plasma concentration of Highly sensitive C-reactive protein (hsCRP)
and circulating AECAs titer measurements. Plasma concentration
of hsCRP was measured using a highly sensitive, automated immunoassay (Roche Diagnosis, Basel, Switzerland). This test provides a
low detection limit of 0.2 mg/l and a variation coefficient of 4.2% in
4 mg/l and 6.3% in 1 mg/l. Each sample was performed in triplicate,
and the mean of the three values was used for the analysis.
Circulating AECAs detection
The autoantibody titer was detected by indirect immunofluorescence using a diagnosis reagent kit from EUROIMMUN (Medizinische Labordiagnostika AG, Luebeck, Germany) with a TITERPLANE technique. This technique provides 100% sensitivity and
specificity for AECAs detection. Cultivated umbilical vein endothelial cells covered the reaction areas of a BIOCHIP. Slides were
incubated with patient’s diluted serum samples. Positive reactions
were marked by granular staining in cytoplasm of human umbilical
vein endothelial cells with fluorescein-labeled antibodies and were
visualised by fluorescence microscopy. (Figure 1). According to the
manufacturers, titers of <1:10 represent the reference range and
the lower detection limit of the test.
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Statistical analysis
Data was processed using the SPSS 15.0 statistical software package (Microsoft). Differences between groups were considered statistically significant for a P<0.05 in two-tailed test. The normality of
continuous variables was analysed using the Kolmogorov-Smirnov
and Shapiro-Wilk tests. The association between categorical variables was studied using the Chi-square test and the Fisher’s Exact
test when required. The association between continuous variables
was analysed using the Mann-Whitney U test and Spearman’s correlation test. Categorical variables were expressed as a percentage and continuous variables as the median (interquartile range
[p25-p75]). All the extreme values and outliers were identified and
double-checked.
RESULTS
Circulating AECAs titer >1:10 was detected in the serum sample of
20 (59%) patients. All the autoantibodies were IgG isotype and all
the serum samples with AECAs titer >1:10 reacted against beta2glycoprotein I, proteinase 3 and collagenase antigens. The baseline
characteristics of the included patients are described in Table 1.
Relationship between circulating AECAs and WBC
There were no statistical differences in lipid profile or WBC count
between the analysed groups (Table 2). However, patients with AECAs titer >1:10 showed a higher blood monocyte count (0.72 [0.601.01] vs. 0.55 [0.45-0.79] X103/µL p=0.03). Eosinophils count was
also higher in patients with AECAs titer >1:10 (0.28 [0.22-0.36] vs.
0.25 [0.07-0.29] X103/µL p=0.05) (Table 2 and figure 2).
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Figure 2: Relationship between white blood cell count (WBC) and anti-endothelial cell antibodies (AECAs).
Monocyte count (a) and eosinophil count (b) were higher in patients with circulating AECAs titer ≥1:10. *Blood cell count units: X103 /µL.
Table 1: Baseline characteristics of the included patients
AECAs titer ≥1:10
% (n=20)
AECAs titer <1:10
% (n=14)
p value
Age and Cardiovascular risk factors
Age (years)
63 [60-68]
67 [57-74]
0.98
Diabetes mellitus
5 (25%)
3 (21%)
1
History of Smoking
10 (50%)
6 (43%)
0.73
Coronary heart disease
4 (20%)
4 (28%)
0.68
Hypertension
11 (55%)
7 (50%)
1
Cerebrovascular disease
2 (10%)
0 (0%)
0.50
Chronic renal failure
2 (10%)
1 (7%)
1
Chronic pulmonary disease
1 (5%)
2 (14%)
0.55
Medical treatment
118
Anti-platelet
15 (75%)
11 (78%)
1
Statins
6 (30%)
4 (28%)
0.61
ACE inhibitors
8 (40%)
5 (36%)
0.54
Nitrates
1 (5%)
2 (14%)
0.55
Beta-blockers
2 (10%)
2 (14%)
1
Calcium antagonist
5 (25%)
4 (28%)
0.56
Beta-agonists
2 (10%)
2 (14%)
1
Oral anticoagulants
0 (0%)
2 (14%)
0.16
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Table 2: Lipid profile and white blood cell count of the included patients
AECAs titer ≥1:10
(n=20)
AECAs titer <1:10
(n=14)
p value
Lipid profile
Cholesterol (mg/dl)
211 [171-251]
203 [151-248]
0.43
Triglycerides (mg/dl)
148 [132-188]
139 [107-167]
0.53
High-density lipoprotein (mg/dl)
45 [38-50]
49 [42-58]
0.53
Low-density lipoprotein (mg/dl)
116 [106-166]
116 [73-173]
0.48
29 [26-38]
28 [21-33]
0.53
Very low-density lipoprotein (mg/dl)
White blood cell count
White blood cell count (X103/µL)
8.26 [5.89-12.32]
7.48 [5.83-10.15]
0.39
Neutrophils (X103/µL)
4.40 [3.63-7.21]
3.75 [2.25-5.49]
0.12
Lymphocytes (X103/µL)
2.62 [1.98-3.61]
1.67 [1.59-3.53]
0.09
Monocytes (X103/µL)
0.72 [0.60-1.01]
0.55 [0.45-0.79]
0.03
Eosinophils (X103/µL)
0.28 [0.22-0.36]
0.25 [0.07-0.29]
0.05
Basophils (X103/µL)
0.06 [0.05-0.08]
0.05 [0.04-0.06]
0.11
Relationship between circulating AECAs and hsCRP
Relationship between hsCRP and blood cell count
We found higher hsCRP levels in patients with circulating AECAs
titer >1:10 (7.60 [4.25-10.20] vs. 4.90 [3.20-7.00] mg/L p=0.02)
(Figure 3).
We did not find any significant statistical correlation between WBC
and hsCRP in our sample.
DISCUSION
Figure 3: Relationship between C-reactive protein (hsCRP)
and anti-endothelial cell antibodies (AECAs). Levels of hsCRP
were higher in subjects with circulating AECAs titer ≥1:10.
According to the findings of this study, the presence of circulating
AECAs in patients with PAD could be associated with the systemic
inflammatory status that surrounds the disease and with high
monocyte and eosinophil blood cell count. Circulating AECAS have
been related to PAD. Subjects seropositive to these autoantibodies presented worse endothelial dysfunction parameters (Flowmediated arterial dilatation), higher levels of inflammation markers
(hsCRP) and higher Intiman-media thickness measurements (18).
This data suggest a possible role of circulating AECAs on the atherogenic process.
Endothelial dysfunction acts as a primary pathogenic event for
atherosclerosis, as it occurs before structural changes are evident
on angiogram or ultrasound scan and it is not correlated with the
disease’s severity (19). The loss of endothelial regulation has been
attributed to a reduction in nitric oxide (NO) bioactivity, and to an
increased oxygen-free radical formation in the context of the proinflammatory status found in the disease (20,21).
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In this context, circulating AECAs may cause endothelial damage
through several mechanisms. Polyclonal IgG fractions of these autoantibodies can mediate endothelial cell lysis when incubated in
vitro with the complement or in the presence of mononuclear cells
to induce an antibody-dependent cellular cytotoxicity (15). Circulating AECAs can also induce endothelial apoptosis by themselves
or together with natural killer cells. Signaling for apoptosis could
be initiated through the interaction of these autoantibodies with
the Fas receptor or with the integrin α3B1 and α6B1-NAG-2 protein
complex (12). Furthermore, some published data have suggested
that circulating AECAs might cause endothelial cell apoptosis
through the NO pathway (22,23).
On the other hand, Inflammation has a key role in every aspect
of the atherosclerotic process. Recent investigations reported
that the presence of high-risk carotid plaques is associated with
increased levels of inflammatory markers, such as hsCRP and leukocyte count (24-26). Another study found that the prevalence of
unstable hypoechoic plaques was higher in PAD than in coronary
artery disease patients, probably because, consistent with previous
studies, the PAD group has more pronounced inflammatory profile
(27). Neutrophil count was associated with a more severe clinical
presentation of atherosclerosis independently of sex, age and classic risk factors (27).
Relationship between circulating AECAs,
monocyte blood cell count and hsCRP levels
In the present research we have observed an association between
AECAs titer ≥1:10 and high monocyte blood cell count in patients
with PAD. These findings are of great interest, considering that
monocytes contribute actively to atherosclerotic lesion development. The infiltration of circulating monocytes into the artery wall
is one of the earliest events in the development of the atherosclerotic lesion. These white blood cells are believed to play a critical
role in the atherosclerosis initiation, plaque instability and artery remodeling stages (28-30). Blood monocytes have been also independently associated with subclinical PAD after adjustment for other
inflammatory markers (4).
In this context, circulating AECAs could activate endothelial cells
through a NF-κB dependent mechanism, leading to the expression
of leukocyte adhesion molecules (intercellular adhesion molecule
1 [ICAM-1], vascular cell adhesion molecule 1 [VCAM-1], E-selectin)
and increasing the release of pro-inflammatory cytokines (interleukin 1 [IL-1], interleukin 6 [IL-6]) in vitro (16,17). These observations
are consistent with the finding of raised hsCRP levels in patients
with PAD and circulating AECAs. The relationship found between
these autoantibodies and hsCRP levels suggest that autoimmunity
could play an important role in the genesis of the chronic inflammation observed in patients with PAD. On the other hand, the
hypothetical endothelial inflammatory chemotaxis enhancement
related to circulating AECAs could justify higher monocyte count
levels when the antibody is present.
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Relationship between circulating AECAs and
eosinophil blood cell count
We also found higher eosinophils count in patients with circulating AECAs. Recent observations suggest that eosinophils may play
a role in coronary atherosclerosis (31). Eosinophils count has been
associated with an increased risk for future cardiovascular events
(32,33). When activated, eosinophils secrete several proteins, among
which eosinophil cationic protein (ECP) stands up (34). This molecule interacts with other immune cells and plasma proteins such as
coagulation factors and the complement system (35). ECP also upregulates endothelial ICAM-1 expression (36) allowing monocytes
adhesion on endothelium. Therefore, eosinophils might cooperate
with, or amplify, circulating AECAs immune mechanisms in mediating endothelial damage.
Our findings do not establish a causal relationship. However, the
observed association of circulating AECAs with monocytes and eosinophils count in patients with PAD supports the hypothesis of an
alleged autoimmune origin of atherosclerosis, given the clear relationship of these blood cells with autoimmune diseases and circulating AECAs pathogenic mechanisms. Although the data obtained
from this study does not have a direct clinical application, its potential benefits lie in the improvement of the etiopathogenic insights
of the atherogenic process.
CONCLUSION
The presence of circulating AECAs in patients with PAD could be associated with the systemic inflammatory status that surrounds the
disease and with high monocyte and eosinophil blood cell count.
However, further prospective studies are required to establish a
causal relationship.
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