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medicina
Article
The Proinflammatory Soluble CD40 Ligand Is Associated with
the Systemic Extent of Stable Atherosclerosis
Tiago Pereira-da-Silva 1,2 ,* , Patrícia Napoleão3, Teresa Pinheiro 4, Mafalda Selas 1, Filipa Silva 1,
Rui Cruz Ferreira 1and Miguel Mota Carmo 5
Citation: Pereira-da-Silva, T.;
Napoleão, P.; Pinheiro, T.; Selas, M.;
Silva, F.; Ferreira, R.C.; Carmo, M.M.
The Proinflammatory Soluble CD40
Ligand Is Associated with the
Systemic Extent of Stable
Atherosclerosis. Medicina 2021,57, 39.
https://doi.org/10.3390/medicina
57010039
Received: 28 November 2020
Accepted: 29 December 2020
Published: 4 January 2021
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4.0/).
1Department of Cardiology, Hospital de Santa Marta, Centro Hospitalar Universitário de Lisboa Central,
1169-024 Lisbon, Portugal; mafalda.selas@gmail.com (M.S.); felipafernandes@gmail.com (F.S.);
cruzferreira@netcabo.pt (R.C.F.)
2NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa,
1169-056 Lisbon, Portugal
3Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa,
1649-028 Lisbon, Portugal; napoleao.patricia@gmail.com
4Instituto de Bioengenharia e Biociências, Departamento de Engenharia e Ciências Nucleares,
Instituto Superior Técnico, Universidade de Lisboa, 2695-066 Lisbon, Portugal;
teresa.pinheiro@tecnico.ulisboa.pt
5Chronic Diseases Research Center (CEDOC), NOVA Medical School, Faculdade de Ciências Médicas,
Universidade NOVA de Lisboa, 1150-082 Lisbon, Portugal; mabmc@sapo.pt
*Correspondence: tiagopsilva@sapo.pt; Tel.: +351-919908505
Abstract:
Background and objectives: Polyvascular atherosclerosis is frequent and associated with a
high cardiovascular risk, although the mechanisms regulating the atherosclerosis extent to single
or multiple arterial territories are still poorly understood. Inflammation regulates atherogenesis
and soluble CD40 ligand (sCD40L) is an inflammatory mediator associated with the presence of
single-territorial atherosclerosis. We assessed whether the sCD40L expression is associated with the
atherosclerosis extent to single or multiple arterial territories and with the atherosclerosis severity
in different territories. Materials and Methods: We prospectively enrolled 94 participants with no
atherosclerosis (controls, n= 26); isolated coronary atherosclerosis (group 1, n= 20); coronary
and lower extremity (LE) atherosclerosis (group 2, n= 18); coronary and carotid atherosclerosis
(group 3, n= 12); and coronary, LE, and carotid atherosclerosis (group 4, n= 18). Serum sCD40L
levels were quantified. Results: The sCD40L levels (ng/mL, mean (standard deviation)) were 4.0
(1.5), 5.6 (2.6), 7.2 (4.2), 5.9 (3.7), and 5.1 (2.4) in controls and groups 1 to 4, respectively (ANOVA
p= 0.012
). In nonrevascularized patients, the sCD40L levels were significantly higher in group 2
than in group 1 and were correlated with the number of LE diseased segments. Prior LE bypass
surgery was associated with lower sCD40L levels. Coexistence of coronary and LE atherosclerosis
was independently associated with the sCD40L levels. Conclusions: The sCD40L levels were increased
in stable atherosclerosis, particularly in polyvascular coronary and LE atherosclerosis. The number
of LE diseased segments and prior LE revascularization were associated with sCD40L expression.
To our knowledge, these are novel data, which provide insights into the mechanisms underlying
multi-territorial atherosclerosis expression. sCD40L may be a promising noninvasive tool for refining
the stratification of the systemic atherosclerotic burden.
Keywords:
atherosclerosis; carotid artery disease; coronary artery disease; inflammation;
lower extremity
arterial disease; soluble CD40 ligand
1. Introduction
Little is known on the mechanisms that regulate the atherosclerosis extent to single
or multiple arterial beds [
1
,
2
]. Specifically, it is not completely understood why some
patients develop isolated coronary artery disease (CAD), while others develop a more
Medicina 2021,57, 39. https://doi.org/10.3390/medicina57010039 https://www.mdpi.com/journal/medicina
Medicina 2021,57, 39 2 of 13
systemic disease involving coronary and extra-coronary lesions [
1
,
2
]. Inflammation regu-
lates atherogenesis and could partially explain the heterogeneity of stable atherosclerosis
presentation [
3
]. The importance of identifying inflammatory mediators associated with
stable atherosclerosis expression is recognized not only for better understanding of the
pathophysiology, but also for clinical practice since they may potentially be used as diag-
nostic biomarkers and therapeutic targets [
4
]. In fact, the study on inflammation recently
regained interest after the groundbreaking results of a pure anti-inflammatory agent in
patients with atherosclerosis, in the Canakinumab Antiinflammatory Thrombosis Outcome
Study [4].
The soluble CD40 ligand (sCD40L) is a proinflammatory mediator that has a mech-
anism of action distinct from other inflammatory mediators [
5
–
8
]. It is released into
circulation mainly by activated platelets and interacts with different cells that participate in
vascular inflammation and atherogenesis, including endothelial cells, macrophages, and T
cells [
5
–
8
]. Furthermore, sCD40L is associated with vascular function [
9
,
10
]. Previous stud-
ies reported increased sCD40L levels in stable atherosclerosis of single territories, including
the coronary [
11
–
13
], lower extremity (LE) [
14
–
16
], and carotid [
17
–
19
] vascular beds. To
the best of our knowledge, there is no published prospective data on a potential association
between sCD40L and disease extent to single or multiple arterial territories. Polyvascular
atherosclerosis warrants special attention not only because it may have a different patho-
physiology from that of single-vascular atherosclerosis, but also because it is frequent
in clinical practice and associated with a higher risk of ischemic events [
20
–
22
]. On the
other hand, the sCD40L levels were reported to be associated with CAD severity [
12
,
13
],
whereas data on the sCD40L expression according to extra-coronary atherosclerosis severity
is very scarce [23,24].
This pilot study aimed to explore whether the serum sCD40L levels are associated
with the extent of stable atherosclerosis to single (coronary) or multiple (coronary and
extra-coronary) arterial territories and with the atherosclerosis severity in extra-coronary
territories. We hypothesized that the sCD40L levels differ according to the systemic extent
of atherosclerosis, specifically in isolated CAD and polyvascular atherosclerosis, and the
severity of atherosclerosis in different territories.
2. Materials and Methods
This study is a part of a project aimed at assessing the mechanisms underlying stable
atherosclerosis expression in single (coronary) and multiple (coronary plus LE and/or
carotid) arterial territories. The study protocol was approved by the ethics committees
of the involved institutions (NOVA Medical School, Faculdade de Ciências Médicas, Uni-
versidade NOVA de Lisboa, Nr. 000176, in 11 November 2015, and Centro Hospitalar
Universitário de Lisboa Central, Nr. 245/2015, in 1 October 2015). The investigation
conformed to the principles outlined in the Declaration of Helsinki. All participants signed
informed consent forms for inclusion before they participated in the study.
2.1. Recruitment of Participants
For assessing the sCD40L expression in atherosclerosis of single and multiple vas-
cular beds, we prospectively recruited five groups of age- and sex-matched participants
followed in our center: controls, with no coronary, LE, or carotid atherosclerosis; group 1,
with isolated CAD; group 2, with coronary and LE atherosclerosis; group 3, with coro-
nary and carotid atherosclerosis; and group 4, with atherosclerosis of the three territories.
All participants were screened for obstructive atherosclerotic disease in the three territories.
CAD was excluded in controls if they presented no effort angina; no evidence of CAD
on coronary computed tomography angiography, including a calcium score of 0 and no
soft plaques; and no positive myocardial stress test (the latter was not mandated to be
assessed as per protocol). In other participants, CAD was defined as luminal stenosis of at
least 50% for the left main artery or at least 70% for other epicardial vessels on invasive
coronary angiography. LE arterial disease was defined as a significant (
≥
50%) stenosis
Medicina 2021,57, 39 3 of 13
on Doppler ultrasound (DUS) at rest [
25
,
26
] or the combination of chronic claudication
and an ankle–brachial index equal to or less than 0.9 [
26
,
27
]. DUS was performed for the
characterization of LE arterial disease. LE arterial disease was excluded in the case of
no significant (
≥
50%) stenosis on DUS or in the case of absent chronic claudication and
an ankle–brachial index higher than 0.9 [
26
,
27
]. Carotid artery disease was defined as
stenosis of at least 50% on DUS and was excluded in the absence of any significant (
≥
50%)
stenosis [
26
,
27
]. The mean and maximal intima–media thickness (IMT) were measured as
previously described in individuals without overt arterial injury (in case of no significant
carotid artery stenosis) [
28
]. All DUS studies of the LE and carotid arteries were performed
according to a standardized protocol, using the GE Logiq S7 Expert Ultrasound System,
and measurements were performed while following published guidelines [26,27,29].
The exclusion criteria were as follows: patients with acute ischemic events within
12 months, either coronary, LE, or cerebrovascular events; those with coronary artery by-
pass grafting (CABG) or LE bypass surgery performed within 12 months; those with prior
carotid endarterectomy or prior percutaneous intervention of the coronary, LE, or carotid
arteries; those with critical limb ischemia (with ischemic rest pain), heart failure, hemo-
dynamically significant valvular heart disease, hematological disorders, active infection,
history of malignancy, chronic kidney disease (stage 4 or 5), or severe hepatic dysfunction;
those under 18 years of age; or those unable or unwilling to consent to study participa-
tion. If performed at least 12 months before inclusion, prior CABG and/or LE bypass
surgery were not exclusion criteria since the presence and extent of atherosclerotic lesions,
which were the focus of this study, are not modified by the surgical placement of bypass
conduits [26,30].
2.2. Sample Size
No previous studies reported data on the sCD40L levels in both single- and multi-
territorial atherosclerosis with the systematic assessment of different territories,
which would be valuable for supporting the sample size estimation in our study. In this
pilot study, we planned to recruit at least 20 controls, 20 patients with isolated CAD (group
1), and 40 patients with coronary and extra-coronary atherosclerosis (groups 2 to 4), in-
cluding at least 10 patients per group in groups 2 to 4. The recruitment of participants for
each group continued even after meeting the minimum number of participants until the
minimum sample size was achieved for all groups.
2.3. Data Collection
Data were collected prospectively after patient inclusion. A standardized record of
clinical, demographic, laboratory, echocardiographic, DUS, computed tomography angiog-
raphy, and invasive coronary angiography data was obtained from each participant. For
evaluating the severity of LE disease, the number of arterial segments with obstructive dis-
ease on both sides was assessed, including the external iliac, common femoral, superficial
femoral, popliteal, anterior tibial, posterior tibial, and fibular arteries [
25
,
26
,
31
]. The LE
lesions were classified as proximal if they were located in the popliteal artery or above or
distal if they were located below [26].
2.4. Blood Sampling and sCD40L Measurements
Peripheral blood was collected early in the morning under fasting conditions.
Serum was separated by centrifugation (500
×
gfor 10 min) within 15 min of sampling.
Aliquots were stored at
−
80
◦
C; samples were thawed only once. The sCD40L levels were
measured in serum by an enzyme-linked immunosorbent assay commercial kit (R&D
Systems, Minneapolis, MN, USA). Each sample was measured in duplicate. The intra-assay
variation among the duplicates for all samples was less than 10%.
Medicina 2021,57, 39 4 of 13
2.5. Statistical Analysis
Discrete variables are presented as frequency (percentage); continuous variables are
presented as mean (standard deviation) in normally distributed data or median (interquar-
tile range) in variables without a normal distribution (Shapiro–Wilk test). Categorical
variables were analyzed using the chi-square or Fisher’s exact tests. Continuous vari-
ables were analyzed using Student’s t-test or the Mann–Whitney test when normality was
not verified. Comparisons between multiple groups were performed using the analysis
of variance (ANOVA) in normally distributed data and Kruskal–Wallis test in variables
without a normal distribution; the Bonferroni post hoc correction was used for multiple
pairwise comparisons. Pearson’s correlation was used to test correlations between con-
tinuous variables. The multivariate linear regression analysis was performed to identify
the independent predictors of the sCD40L levels, among all available data. Outliers were
excluded, as appropriate [
32
]. Considering the association between prior CABG or LE
bypass surgery and lower sCD40L levels in our sample, we performed a post hoc analysis
to explore whether the sCD40L levels varied with the atherosclerosis extent to single or
multiple arterial territories and with the atherosclerosis severity in extra-coronary territo-
ries, excluding patients with prior revascularization. The level of significance considered
was α= 0.05. Analyses were conducted using the SPSS software, version 26 (IBM).
3. Results
3.1. Clinical Characteristics, Laboratory Results, and Atherosclerosis Data of Participants
A total of 94 participants were included: 26 controls, 20 with isolated CAD (group 1),
18 with coronary and LE disease (group 2), 12 with coronary and carotid disease (group 3),
and 18 with disease of the three territories (group 4). Clinical characteristics, laboratory
results, and atherosclerosis data of participants are presented in Table 1. The differences in
clinical characteristics and laboratory data across groups were driven by controls, where
hypertension, dyslipidemia, diabetes mellitus, smoking history, and the use of antiplatelet
and statin therapy were less prevalent; the neutrophil count, neutrophil/lymphocyte ratio,
and creatinine levels were lower; and the high-density lipoprotein cholesterol levels were
higher than those in patients with atherosclerosis. The distribution of these parameters did
not differ across groups 1 to 4.
Regarding atherosclerosis data, the distribution of CAD parameters did not differ
significantly across groups 1 to 4, including the number of vessels with obstructive disease,
number of obstructive lesions, Gensini score, or rates of prior CABG.
Among patients with LE atherosclerosis (groups 2 and 4), group 4 showed a higher
prevalence of bilateral LE disease and higher number of LE arterial segments with obstruc-
tive disease. The presence of proximal LE lesions and rates of prior LE bypass surgery did
not differ between groups 2 and 4, although the revascularization rates were nonsignifi-
cantly higher in the latter (11.1% vs. 33.3%, respectively, p= 0.109).
Regarding patients with carotid artery disease (groups 3 and 4), there were no dif-
ferences between the two groups regarding the rates of bilateral carotid artery disease.
The maximal and mean IMT did not differ across controls and groups 1 and 2.
Medicina 2021,57, 39 5 of 13
Table 1. Characteristics of the participants according to the involved territories of atherosclerosis.
Controls Group 1 Group 2 Group 3 Group 4 p-Value
Territories of atherosclerosis None Coronary Coronary + LE Coronary + Carotid
Coronary + LE + Carotid
n26 20 18 12 18
Clinical characteristics
Age, years 59 (53–69) 65 (56–70) 67 (57–72) 59 (51–73) 69 (60–75) 0.079
Male, n(%) 23 (88.5) 18 (90.0) 16 (88.9) 10 (83.3) 17 (94.4) 0.912
Hypertension, n(%) 14 (53.8) 17 (85.0) 118 (100.0) 111 (91.7) 118 (100.0) 1<0.001
Dyslipidemia, n(%) 18 (69.2) 19 (95.0) 118 (100.0) 111 (91.7) 17 (94.4) 10.010
Diabetes mellitus, n(%) 3 (11.5) 6 (30.0) 8 (44.4) 16 (50.0) 19 (50.0) 10.036
Smoking history, n(%) 6 (23.1) 9 (45.0) 12 (66.7) 14 (33.3) 12 (66.7) 10.014
LVEF > 50%, n(%) 26 (100.0) 20 (100.0) 18 (100.0) 12 (100.0) 18 (100.0) –
Antiplatelet agent, n(%) 6 (23.1) 20 (100.0) 117 (94.4) 111 (91.7) 118 (100) 1<0.001
Statin therapy, n(%) 13 (50.0) 18 (90.0) 116 (94.1) 111 (91.7) 116 (88.9) 10.001
Laboratory parameters
Hemoglobin, g/dL 13.9 (12.9–15.0) 14.53 (10.0–15.1) 14.1 (13.2–14.6) 12.0 (11.4–13.4) 212.9 (12.1–14.2) 0.017
Leukocyte count, 109/L 6.4 (1.7) 7.4 (1.9) 7.3 (1.7) 7.5 (2.2) 8.1 (1.7) 0.080
Neutrophil count, 109/L 3.2 (2.5–4.8) 4.1 (3.4–5.2) 3.9 (3.4–4.8) 4.0 (3.4–6.7) 4.7 (3.6–6.0) 10.043
Lymphocyte count, 109/L 1.9 (1.7–2.2) 2.1 (1.6–2.4) 2.1 (1.6–2.8) 1.7 (1.2–2.3) 2.2 (1.6–2.6) 0.401
Neutrophil/lymphocyte ratio 1.9 (0.7) 2.3 (1.1) 2.1 (1.0) 2.9 (1.1) 12.4 (1.0) 0.026
Platelet count, 109/L 242 (191–274) 209 (176–269) 219 (195–264) 229 (137–251) 227 (203–263) 0.854
Fasting glycaemia, mg/dL 89 (80–98) 94 (86–129) 94 (83–125) 99 (84–157) 85 (75–123) 0.385
Percentage of glycosylated hemoglobin 5.6 (5.2–5.9) 5.9 (5.6–6.7) 5.9 (5.5–6.1) 5.8 (5.4–7.4) 5.9 (5.3–7.7) 0.185
Creatinine, mg/dL 0.8 (0.7–0.9) 0.9 (0.8–1.1) 0.8 (0.8–1.2) 0.9 (0.8–1.4) 1.1 (0.9–1.5) 10.002
Total cholesterol, mg/dL 186 (51) 164 (38) 172 (50) 153 (50) 173 (49) 0.329
LDL-cholesterol, mg/dL 99 (77–141) 95.0 (71–120) 106 (83–120) 65 (56–132) 117 (82–142) 0.297
HDL-cholesterol, mg/dL 51 (44–58) 35.0 (31–41) 135 (31–45) 140 (27–44) 140 (32–42) 1<0.001
Triglycerides, mg/dL 106 (67–144) 142 (98–206) 115 (83–204) 100 (62–177) 117 (95–171) 0.423
C-reactive protein, mg/L 4.1 (2.0) 3.7 (1.4) 3.8 (1.1) 4.1 (2.3) 3.3 (2.0) 0.151
Coronary artery disease
Nr. of vessels with obstructive disease *
0 (0–0) 3 (2–3) 13 (2–4) 13 (3–3) 13 (2–4) 1<0.001
Nr. of obstructive lesions 0 (0–0) 4 (2–5) 14 (3–5) 14 (3–5) 14 (3–5) 1<0.001
Gensini score 0 (0–0) 81 (41–97) 183 (42–118) 148 (40–116) 143 (36–87) 1<0.001
Prior CABG, n(%) 0 (0.0) 7 (35.0) 17 (38.9) 16 (50.0) 13 (16.7) 10.002
Medicina 2021,57, 39 6 of 13
Table 1. Cont.
Controls Group 1 Group 2 Group 3 Group 4 p-Value
LE arterial disease
Bilateral disease, n(%) 0 (0.0) 0 (0.0) 10 (55.5) 1,2,4,5 0 (0.0) 15 (83.3) 1–4 <0.001
Any proximal lesion, n(%) 0 (0.0) 0 (0.0) 10 (55.5) 1,2,4 0 (0.0) 12 (66.7) 1,2,4 <0.001
Nr. of segments with obstructive
disease 0 (0–0) 0 (0–0) 2 (1–4) 1,2,4,5 0 (0–0) 4 (3–5) 1–4 <0.001
Prior bypass surgery, n(%) 0 (0.0) 0 (0.0) 2 (11.1) 1,2,4 0 (0.0) 6 (33.3) 1,2,4 <0.001
Carotid artery disease
Bilateral disease, n(%) 0 (0.0) 0 (0.0) 0 (0.0) 3 (25.0) 1–3 8 (44.4) 1–3 <0.001
Mean IMT, mm 0.68 (0.11) 0.67 (0.14) 0.76 (0.06) - - 0.296
Maximal IMT, mm 0.80 (0.13) 0.86 (0.18) 0.94 (0.09) - - 0.105
Categorical variables are expressed as frequency (percentage) and continuous variables as mean (standard deviation) or median (interquartile range). CABG—coronary artery bypass grafting; HDL—high-density
lipoproteins; IMT—intima–media thickness; LDL—low-density lipoproteins; LE—lower extremity; LVEF—left ventricular ejection fraction; Nr.—number.
1
p-Value < 0.05 vs. controls;
2
p-Value < 0.05 vs. group 1;
3
p-Value < 0.05 vs. group 2;
4
p-Value < 0.05 vs. group 3;
5
p-Value < 0.05 vs. group 4; * for the assessment of the number of vessels with obstructive disease, the left main, left anterior descending, circumflex,
and right coronary arteries were considered separately, with a total score ranging from 0 to 4.
Medicina 2021,57, 39 7 of 13
3.2. Variation of the sCD40L Levels According to the Systemic Extent of Atherosclerosis
The sCD40L levels differed across groups (ANOVA p = 0.012) (Figure 1a). Patients
from groups 1 (isolated CAD) and 2 (coronary and LE disease) showed significantly higher
sCD40L levels than controls, and those from groups 3 (coronary and carotid disease) and 4
(disease of the three territories) showed nonsignificantly higher sCD40L levels than controls.
Excluding patients with prior CABG and/or LE bypass surgery, the sCD40L levels were
significantly higher in patients from group 2 (coronary and LE disease) compared with
those in patients from group 1 (isolated CAD) (Figure 1b).
Figure 1.
Soluble CD40 ligand levels according to the presence of atherosclerosis in different arterial territories. (
a
) in the
whole sample, and (
b
) excluding patients with prior revascularization of the coronary and/or lower extremity arterial
territories. Soluble CD40 ligand values are expressed as mean (standard deviation). sCD40L—soluble CD40 ligand.
*p-Value < 0.05 vs. controls; ¥ p-Value < 0.05 vs. isolated coronary artery disease.
3.3. Variation of the sCD40L Levels According to the Severity of Atherosclerosis in
Extra-Coronary Territories
For the LE disease, no significant correlation was found between the sCD40L levels
and number of arterial segments with obstructive disease (r = 0.157, p= 0.147). However,
a weak positive correlation between the sCD40L levels and number of arterial segments
with obstructive disease was observed excluding patients with prior LE bypass surgery
(
r = 0.238,
p= 0.034) and those with prior CABG and/or LE bypass surgery (r = 0.281,
p= 0.027
). The median number of LE segments with obstructive disease was 3 (interquartile
range 2–5); classifying patients with LE disease according to the median number of diseased
segments, the sCD40L levels were significantly higher in patients with three or more LE
diseased segments, but not in patients with less than three, compared with those with
no LE disease (Figure 2). sCD40L did not differ according to the presence of bilateral or
proximal LE disease (Supplementary Materials, Table S1).
Medicina 2021,57, 39 8 of 13
Figure 2.
Soluble CD40 ligand levels according to the number of segments of the lower extremity with
obstructive disease in patients without prior lower extremity bypass surgery. Soluble CD40 ligand
values are expressed as median (interquartile range). sCD40L—soluble CD40 ligand.
*p-Value < 0.05
vs. no segments with obstructive disease.
Of note, among patients with LE disease, prior LE bypass surgery was associated with
lower sCD40L levels (Figure 3). The median time elapsed from LE bypass surgery was
4 years (interquartile range 2–9 years) (Supplementary Materials, Table S2). There were no
differences between patients with and without prior LE bypass surgery regarding clinical
characteristics, other laboratory data, CAD severity, rates of prior CABG, or proportion of
bilateral or of proximal LE disease; there was a trend for a higher number of LE diseased
segments in patients with prior LE bypass surgery (Supplementary Materials, Table S2).
Figure 3.
Soluble CD40 ligand levels in patients with lower extremity atherosclerosis with and
without prior lower extremity bypass surgery. Soluble CD40 ligand values are expressed as mean
(standard deviation). sCD40L—soluble CD40 ligand.
Medicina 2021,57, 39 9 of 13
For carotid artery disease, the sCD40L levels showed no association with the presence
of bilateral disease and no correlation with the mean or maximal IMT (Supplementary
Materials, Table S1). Furthermore, no associations were found after excluding patients with
prior CABG and/or LE bypass surgery (data not shown).
3.4. Predictors of the sCD40L Levels
A detailed univariate analysis on parameters associated with the sCD40L levels is
presented in Table S1 (Supplementary Materials) and a stratified analysis by study group
is presented in Table S3 (Supplementary Materials). In the multivariate linear regression
analysis, the independent predictors of the sCD40L levels were coexistent coronary and LE
obstructive atherosclerosis, prior CABG, and leukocyte count (Table 2).
Table 2.
Independent predictors of the soluble CD40 ligand levels by multivariate linear regres-
sion analysis.
Predictors β95% CI p-Value
Coexistent coronary and lower extremity
atherosclerosis 2.512 1.054 to 3.970 0.001
Prior coronary artery bypass grafting −1.758 −3.159 to −0.357 0.015
Leukocyte count 0.417 0.102 to 0.732 0.010
95% CI—95% confidence interval.
4. Discussion
In this prospective case–control study, three main findings stood out: the sCD40L
levels varied according to the systemic extent of atherosclerosis to single or multiple arterial
territories, the sCD40L levels were associated with the extent of atherosclerosis in the LE
territory, and prior LE bypass surgery was associated with lower sCD40L levels among
patients with LE atherosclerosis.
To the best of our knowledge, we present the first prospective study assessing sCD40L
expression in single- and polyvascular atherosclerotic disease, including three major terri-
tories of atherosclerosis. sCD40L increased from controls to patients with isolated CAD
and further to patients with combined CAD and LE disease (excluding those with prior
revascularization). Investigation addressing inflammation in polyvascular atherosclero-
sis is relevant since it may have a distinct pathophysiology from that of single-vascular
atherosclerosis and is a common clinical scenario associated with a higher risk morbidity
and mortality [
20
–
22
]. Some inflammatory parameters have been associated with the
presence of atherosclerosis in specific arterial territories and with atherosclerosis sever-
ity within each territory. However, data on the inflammatory signature in single- and
multi-territorial atherosclerosis, with a prospective and systematic assessment of different
territories, including the coronary, LE, and carotid artery territories, are very scarce [
33
].
Specifically, for sCD40L, which has a distinct mechanism of action compared with other
inflammatory markers [
5
–
8
], such data are not available. Two studies reporting on the
sCD40L levels in patients with stable atherosclerosis included a subgroup with polyvascu-
lar atherosclerosis but the sCD40L levels were not specifically reported in patients with
single- and polyvascular disease [
34
,
35
]. In a study on patients with LE atherosclerosis,
the sCD40L levels were specifically reported in a subgroup with coexistent CAD; however,
not all patients with LE atherosclerosis were screened for CAD in this retrospective study,
which limits the interpretation of the results [
23
]. In our study, the higher sCD40L levels in
atherosclerosis of multiple (coronary and LE) territories suggest that sCD40L is a common
denominator of the atherosclerosis expression.
The sCD40L levels were higher in combined CAD and LE atherosclerosis, but not in
combined CAD and carotid artery disease, compared with CAD alone. We speculate that
the regulation of the sCD40L levels may be mainly associated with the presence of CAD and
the coexistence of carotid artery disease may not impact further on the sCD40L levels, on
the contrary to LE disease. The local expression of inflammatory markers differs in carotid
Medicina 2021,57, 39 10 of 13
and femoral atherosclerotic plaques [
36
], and the stimulated LE iliac arteries may express
more intensively CD40L in situ than the stimulated carotid arteries [
37
]. On the other hand,
obstructive atherosclerosis of the LE could result in a higher degree of oxidative stress and
inflammation compared with carotid artery disease considering the highly demanding
LE muscles during physical effort and bilateral carotid blood supply to the cerebral terri-
tory [
38
]. For instance, the expression of miR-210, an adaptive microRNA to oxidative stress
and inflammation, is altered in the presence of LE atherosclerosis but not in carotid artery
disease [
38
]. This could explain the higher levels of the proinflammatory sCD40L in the
presence of LE atherosclerosis compared with carotid atherosclerosis, in patients with CAD.
Of note, the sCD40L levels were nonsignificantly higher in group 4 (atherosclerosis of the
three arterial territories) compared with controls, corresponding to a trend consistent with
the results of increased sCD40L levels in group 2 (atherosclerosis of the coronary and LE
territories) compared with controls. On the other hand, group 4 presented similar sCD40L
levels compared with group 1 (CAD alone). This finding is difficult to explain based on the
extensive post hoc analysis performed. Nevertheless, group 4 presented a slightly better
metabolic control compared with group 2, as reflected by nonsignificant lower fasting
glycemia levels, higher HDL levels (which inhibit platelet activity through scavenger re-
ceptor B type I), and lower serum triglyceride levels, which may have contributed to lower
sCD40L levels in group 4 [
39
–
43
]. We acknowledge that possible unmeasured confounders
may have contributed to lower sCD40L levels in group 4.
The second main finding of this study was the association between the sCD40L levels
and atherosclerosis extent within the LE territory. These results are consistent with the very
few studies describing the sCD40L levels according to the severity of LE disease, assessed
by the lesion length [
23
] or by an angiographic score based on the degree of luminal stenosis
in each arterial segment [
31
]. For the carotid arteries, the association between the sCD40L
levels and atherosclerosis severity is less well established [24].
Third, we observed that the sCD40L levels were lower in patients with prior LE
bypass surgery. To our knowledge, this finding has not been reported. We speculate that
the association between the proinflammatory sCD40L and atherosclerosis is bidirectional,
with inflammation contributing to atherogenesis, as demonstrated in animal models [
44
],
and with ischemia driven by atherosclerosis exacerbating inflammation [
35
]. In our sample,
prior revascularization may have reduced ischemia, without modifying atherosclerosis
burden of native arteries (since no endarterectomy was performed), which in turn may
have reduced the release of reactive oxygen species, cytokines, and other inflammatory
mediators, including sCD40L [
26
,
45
]. Consistently, the sCD40L levels were lower in pa-
tients with prior CABG compared with those without, among patients with CAD in this
sample [
12
]. Immediately after CABG and LE surgical revascularization, an increase in
the sCD40L levels has been reported, probably related to the surgical procedure [
46
,
47
].
The lower levels of sCD40L that we observed in the long term after LE revascularization
is a novel finding, which may add to the knowledge on the pathophysiology of revas-
cularization procedures and inflammation in stable atherosclerosis. Of note, although
the number of participants with prior LE bypass surgery was small, this was a post hoc,
hypothesis-generating analysis. Further studies are needed to confirm this hypothesis.
Finally, in the multivariate analysis, coexistent coronary and LE atherosclerosis and
higher leukocyte count were independently associated with increased sCD40L levels, while
prior CABG was associated with lower sCD40L levels. These data add consistency to the
results and confirm the independent association between inflammation, disease extent,
and ischemic burden.
This study has strengths that should be acknowledged. To our knowledge, we describe
for the first time the variation of the sCD40L expression according to single- and multi-
territorial atherosclerosis, including coronary, LE, and carotid atherosclerosis. The prospec-
tive nature of the study, the systematic screening of the three arterial territories and,
importantly, the multivariate analysis carried out have contributed to higher consistency of
our findings. In addition, analyses on the severity/extent of atherosclerosis in different
Medicina 2021,57, 39 11 of 13
territories added further consistency to the results. Finally, to the best of our knowledge,
the association between prior LE surgical revascularization and lower sCD40L levels has
not been reported.
Our study has some limitations. The sample may be of limited size; however, this study
is pioneer in investigating the variation of sCD40L in single- and multi-territorial disease,
and no data were available to support the sample size estimation. In this exploratory
pilot study, the sample size was enough to detect differences in the sCD40L levels in
atherosclerosis of single and multiple territories. On the other hand, as this is a single-
center study, the results may not be applicable to different settings.
5. Conclusions
The sCD40L levels were higher in patients with atherosclerosis, particularly in those
with polyvascular disease involving CAD and LE disease. The sCD40L levels increased
with higher severity of LE atherosclerosis, assessed by the number of diseased segments,
whereas prior LE surgical revascularization was associated with lower sCD40L levels.
Our results provide insights into the pathophysiology of polyvascular atherosclerosis.
In addition, sCD40L seems to be a promising noninvasive tool for refining the stratification
of the systemic atherosclerotic burden and therefore could contribute to the tailoring of the
primary prevention strategies. These fields deserve future research.
Supplementary Materials:
The following are available online at https://www.mdpi.com/1010-6
60X/57/1/39/s1, Table S1: Association of soluble CD40 ligand levels with clinical characteristics,
laboratory results, and atherosclerosis data, Table S2: Characteristics of patients with lower extremity
atherosclerosis with and without prior lower extremity bypass surgery, Table S3: Association of
soluble CD40 ligand levels with clinical characteristics and laboratory results, stratified by the study
group.
Author Contributions:
Conceptualization, T.P.-d.-S., P.N. and M.M.C.; methodology, P.N. and T.P.;
validation, P.N. and T.P.; formal analysis, T.P.-d.-S. and P.N.; investigation, T.P.-d.-S., P.N., T.P.,
M.S. and F.S.; resources, M.S. and F.S.; data curation, T.P.-d.-S., M.S. and F.S.; writing—original draft
preparation, T.P.-d.-S. and P.N.; writing—review and editing, T.P.-d.-S., P.N., T.P., M.S., F.S., R.C.F. and
M.M.C.; visualization, T.P.-d.-S.; supervision, P.N., R.C.F. and M.M.C.; project administration, P.N. and
M.M.C.; funding acquisition, T.P. All authors have read and agreed to the published version of
the manuscript.
Funding:
T.P. has received research support from Fundação para a Ciência e Tecnologia, Portu-
gal [Project UID/BIO/04565/2020], and Programa Operacional Regional de Lisboa 2020 [Project
N. 007317].
Institutional Review Board Statement:
The study was conducted according to the guidelines of
the Declaration of Helsinki, and approved by the ethics committees of the involved institutions
(NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Nr. 000176,
in 11 November 2015, and Centro Hospitalar Universitário de Lisboa Central, Nr. 245/2015,
in 1 October 2015).
Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author. The data are not publicly available due to personal data protection.
Acknowledgments:
This study is part of the PhD thesis program of one of the authors (T.P.-d.-S.),
supervised (M.M.C.) and co-supervised (P.N.) by other two, conducted in NOVA Medical School,
Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisbon, Portugal. The authors are
grateful to Joana Castro from Medinres—Medical Information and Research, for her advice in the
statistical analysis.
Conflicts of Interest: The authors declare no conflict of interest.
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