IL-6, PF-4, sCD40 L, and homocysteine are associated with the radiological progression of cerebral small-vessel disease: a 2-year follow-up study
Clinical Interventions in Aging
Il-6, PF-4, sCD40 l, and homocysteine are associated with the radiological progression of cerebral small-vessel disease: a 2-year follow-up study
Jacek staszewski 1
Renata Piusińska-Macoch 1
Bogdan Brodacki 1
ewa skrobowska 0
Adam Stępień 1
0 Department of Radiology, Military Institute of Medicine , Warsaw , Poland
1 Clinic of n eurology
8 1 0 2 - l u J - 2 1 n o 0 . 5 1 . 5 3 1 . 5 y b / m o c . s s e r p .voed l.yon PowerdbyTCPDF(ww.tcpdf.org) Background: Endothelial dysfunction (ED) is involved in the pathogenesis of cerebral small vessel disease (SVD), however, it is not clear if specific biomarkers related to ED are associated with radiological progression of SVD. Methods: A single-center, prospective cohort study was conducted among consecutive, adult patients with SVD. Logistic regression was used to analyze the association of each baseline biomarker (highest vs lowest tertile) and the MRI radiological outcome after 2 years. The mean Z-score for vascular inflammation (VI) combined soluble intercellular cell adhesion molecule-1 (sICAM-1), soluble platelet selectin (sP-selectin), CD40 ligand (sCD40 L), platelet factor-4 (PF-4) and homocysteine; Z-score for systemic inflammation (SI) combined high-sensitivity C-reactive protein (hsCRP), interleukin-1α and -6 (IL-1α and IL-6, respectively) and tumor necrosis factor-α (TNF-α). Results: The study group comprised 123 patients (age, mean±SD: 72.2±8 years, 49% females), with lacunar stroke (n=49), vascular dementia (n=48), and vascular parkinsonism (n=26). Moreover, 34.9% patients experienced radiological progression, 43% had progression of isolated white matter lesions (WMLs), 23.2% had new lacunes, and 34.8% had both WMLs progression and new lacunes. After adjustment for confounders (age, sex, blood pressure, MRI lesions load), the PF-4 (OR; 95% CI 5.5; 1.5-21), sCD40L (4.6; 1.1-18.6), IL-6 (7.4; 1.48-37), Z-score for VI (4.5; 1.1-18.6), and, marginally, homocysteine (4.1; 0.99-17) were associated with the risk of any radiological progression; further, homocysteine (2.4; 1.4-14), Z-score for SI (2.1; 1.2-14) and, marginally, IL-6 (6.0; 0.95 -38) were related to the development of new lacunes; PF-4 (7.9; 1.6-38) and, marginally, the Z-score for VI (4.2; 0.9-19.5) were correlated with the risk of WMLs progression. Additional adjustment for clinical SVD manifestations did not significantly alter the results. Conclusion: The data supports the concept that ED modulates the radiological progression of SVD and WMLs and lacunes are associated with different inflammatory markers.
changes frequently progress over time, resulting in clinical
deterioration. Recent studies have provided evidence that the
disease is mostly non-atheromatous because the vascular risk
factors can explain only a minority of the variance among
the radiological features.3 Endothelial dysfunction (ED) is
currently considered the most important mechanism of the
structural and functional brain-vessel alterations related to
SVD. The course and predictors of radiological progression
in symptomatic SVD, especially in the rarely studied VaP
or VaD, are not well established. We have recently found a
relationship among hemostatic markers – namely, fibrinogen,
tissue factor, and beta-thromboglobulin – and the risk of
radiological progression in patients with LS, VaD, and VaP.4
Considering that ED is involved in the pathogenesis of SVD,
we hypothesized that some blood-derived markers of ED and
the inflammatory cascade are linked to the risk of
radiological progression in patients older than 60 years with different
ww se Materials and methods
:s na A single-center, prospective cohort study, with a 2-year
th rse radiological follow-up, was conducted as a part of the
SHEFfrodm ropF SVD Study.5 The detailed protocol and methods have been
ed thoroughly described elsewhere. Briefly, the study group
laon comprised 123 consecutive patients .60 years who were
odw enrolled between December 20
11 and June 2014
: 49 had
ingg recent LS (39.8%), 48 had VaD (39%), and 26 had VaP
inA (21.1%) – all were considered a result of sporadic SVD and
itsonne cdriiatgenrioas(eLdSa,caccocrodridnigngtotoththeetOypxifcoarldrsahdirioelCogoimcaml uannidtycSlitnriockael
trvne Project; VaP and VaD according to the Hurtig or the
InterlIca national Workshop of the National Institute of Neurological
liinC Disorders and Stroke and the Association Internationale
pour la Recherche et l’Enseignement en Neurosciences
(NINDS-AIREN) criteria).6–8 Patients with an acute or
chronic infectious process, malignancy, rheumatologic
disease, significant stenosis ($50%) of a major extracranial
or intracranial artery, atrial fibrillation, non–SVD-related
WMLs, life expectancy of ,6 months, and MRI
contraindications were excluded.
Based on a priori knowledge of biological relevance,
we investigated a set of markers related to ED and systemic
inflammation (SI): high-sensitivity C-reactive protein
(hsCRP), interleukin-1 alpha and -6 (IL-1α and IL-6,
respectively), tumor necrosis factor-α (TNF-α); and ED and
vascular inflammation (VI): soluble intercellular cell adhesion
molecule-1 (sICAM-1), soluble platelet selectin (sP-selectin),
CD40 ligand (sCD40 L), platelet factor-4 (PF-4), and
homocysteine.9 Plasma was isolated from overnight fasting blood
samples, and biomarkers were assessed using commercially
available ELISA kits according to the manufacturer’s protocol
(Bio-Source Europe, Nivelles, Belgium); duplicate readings
were recorded, and the intra-assay coefficients of variation
were ,10%. All assays were conducted by individuals
blinded to the study status. In this article, when a compound
or metabolite is mentioned, it is the serum or plasma
concentration that is stated. To prevent any confounding from
the hyperacute-phase responses, all LS patients underwent
study procedures at least 2 weeks (mean 17.5±3.8 days) after
their index strokes.
MRI examinations were conducted at baseline and after
2 years (mean 22.7±3.7 months; LS 22.6±3.8; VaP 21.7±4.3;
and VaD 23.2±3.3 months, p=0.2), and MRI findings were
categorized according to the Standards for Reporting Vascular
Changes on Neuroimaging (STRIVE) guidelines as the
reference standard.10 Images were rated for the presence of LIs,
periventricular (pWMLs) or deep WMLs (dWMLs), MBs, and
PVS according to the recommended visual SVD and Fazekas
scales.11 Mild WMLs (Grade 1) were defined as punctate
lesions in the deep white matter, with a maximum diameter
of 9 mm for a single lesion and 20 mm for grouped lesions.
Moderate WMLs (Grade 2) were early confluent lesions of 10
to 20 mm single lesions as well as grouped lesions of any
diameter measuring .20 mm with nothing more than connecting
bridges between the individual lesions. Severe WMLs (Grade
3) were single lesions or confluent areas of hyperintensity of
$20 mm of any diameter. All subjects had at least Fazekas
Grade 1 WMLs. One point was awarded on the SVD scale if
(early) there were confluent deep WMLs (Fazekas scores 2
and 3) or irregular periventricular hyperintensities extending
into the deep white matter (Fazekas Score 3). A point was
awarded if moderate to extensive (10–25 or .25) enlarged
PVS were present. Finally, the presence of each of the above
markers produced a minimum score of 0 and a maximum of
4 – representing the total MRI load of SVD. Radiological
progression was defined as an increase in WMLs (WML
Notes: Values are mean±sD; Z-score for VI combined (sICAM-1, sCD40 L, sP-selectin, homocysteine, and PF-4); Z-score for SI combined (hsCRP, IL-1α, Il-6, and TnF- α).
*Compared to patients without radiological progression.
Abbreviations: WMLs, white matter lesions; IL-1α, interleukin 1; Il-6, interleukin-6; TnF, tumor necrosis factor; hsCrP, high-sensitivity C-reactive protein; Pgl, plasma
glucose level; sCD40 L, soluble CD40 ligand; VI, vascular inflammation; SI, systemic inflammation; sICAM, soluble intercellular cell adhesion molecule-1; PF-4, platelet factor-4.
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Notes: Values are mean (±SD) or numbers of patients (%); *Mann–Whitney U test or chi-square test.
Abbreviations: SVD, small-vessel disease; VaD, vascular dementia; VaP, vascular parkinsonism; LS, lacunar stroke; BMI, body mass index; WMLs, white matter lesions;
pWMLs, periventricular WMLs; dWMLs, deep WMLs; PVS, enlarged perivascular spaces; MAP, mean arterial pressure.
progression group) or the development of new lacunes (new
lacunes group) in one or more periventricular and/or
subcortical regions (Figure 1).
Detailed neurological examinations and
neuropsychological evaluation were undertaken at the baseline and follow-up
visits. All patients received optimal medical treatment,
according to guidelines. Baseline risk factors including the
average 24-hour mean arterial pressure (MAP) recorded by
24-hour ambulatory blood pressure monitoring (Schiller
MT-300) and the concentrations of biochemical compounds
were compared between patients with and without
radiological progression. To avoid the problem of multiple
comparisons, we used composite measures. For each patient, the mean
Z-score combined blood markers specific for vascular or
systemic inflammation. A Z-score was calculated as the (value in
an individual minus the mean value in the study population)/
standard deviation. We used logistic regression to obtain the
OR (95% CI) for the association of each biomarker
(highest vs lowest tertile) and each outcome (any progression of
WMLs and/or development of new lacunes, development
The study population comprised 123 older adults (age,
mean±SD, 72.2±8 years) of both sexes (48.8% females)
with symptomatic SVD. Patients with VaD, VaP, and LS
differed in regard to their mean age (74.2±7.9 vs 73±5.9
vs 69.6±8.6, respectively, ANOVA p=0.01), the results of
the SVD scale (1.68±0.8; 1.9±1; and 2.8±0.9; p=0.01), and
female sex distribution (75% vs 34.6 vs 30.6%, χ2 p=0.01);
however, there were no other discrepancies among the
prevalence values of vascular risk factors nor among the
levels of the studied biomarkers. Forty-three patients (35%)
experienced radiological progression; the evolution of WMLs
was observed in 43% (n=18) of these cases, new lacunes in
23.2% (n=10), and both WML progression and new lacunes
in 34.8% (n=15). Among patients who had radiological
progression, 21 patients (48.8%) had clinically evident LS,
17 patients (39.5%) experienced deterioration in cognitive
function, and all subjects had either relevant LI or WML
progression on imaging. Although the mean SVD scores
were similar, more than half of the cases that progressed had
a higher prevalence of dWMLs or VaD (Table 1). No other
variations in the distribution of vascular risk factors were
found. Patients with any level of radiological progression
had elevated levels of homocysteine, sCD40 L, PF-4, and
IL-6, which resulted in higher Z-scores for VI and SI; patients
with isolated new lacunes had elevated homocysteine, IL-6,
and Z-scores for SI; and those with WML progression had
elevated PF-4 and Z-scores for VI compared to patients
without progression (Table 2).
After adjusting for confounders (baseline MAP, SVD
score, age, and sex) in the multivariate analysis, the PF-4,
sCD40 L, IL-6, Z-score for VI and, marginally,
homocysteine, and Z-score for SI were associated with the risk
of any radiological progression; homocysteine, Z-score for
SI and, marginally, IL-6 were related to the development of
new lacunes, and PF-4 and marginally Z-score for VI were
correlated with the risk of WML progression (Table 3).
Additional adjustment for clinical SVD manifestations did
not significantly alter the abovementioned findings.
Our study demonstrated that both WMLs and lacunes
progressed over 2 years, according to the follow-up data, in more
than one-third of older adults with symptomatic SVD. The
principal finding was that, in a panel of different biomarkers,
PF-4, sCD40 L, homocysteine, and IL-6 were each associated
with any level of radiological progression, independent of
age, sex, baseline blood pressure, and MRI markers of SVD.
However, the strength of the associations differed among the
radiological determinants of SVD. Higher levels of
homocysteine and, marginally, of IL-6 predicted the development
of new lacunes only, and WML progression was associated
with elevated PF-4. Although both Z-scores were associated
with any radiological progression in univariate analysis, after
adjusting for confounders, the Z-score representing systemic
inflammation markers was significantly associated only with
the risk of new lacunes, whereas the Z-score for vascular
inflammation was merely marginally related to the risk of
WML progression. Interestingly, these associations did not
change after adjusting for SVD clinical manifestations.
Several longitudinal studies documented that the most
consistent predictor for progression of WMLs is the
baseline severity of WMLs and that radiological markers have
better predictive value with regard to long-term functional
outcome as compared to plasma markers.12 Furthermore,
a meta-analysis and systematic review of 46 prospective
studies revealed the doubled risk of death among patients with
WMLs in comparison to those without WMLs.13 However,
only limited data are available on the relationship between
biomarkers and the radiological progression of SVD, which
results in our data being more important. Our results
supported those from the SMART-MR study, which found an
association between homocysteine levels and radiological
progression. In contrast, some studies found associations
between serum ICAM-1 and CRP levels, whereas – in a 3C
Dijon study – no significant relations between baseline CRP
or IL-6 levels and MRI changes were observed. However,
these studies included mainly asymptomatic patients and
had longer follow-up periods (4–6 years).14–16 The
association among PF-4, sCD40 L, and radiological progression is
a new finding, and, to our knowledge, this association has
not been previously reported. Moreover, the relationship
between plasma markers and clinical progression in SVD is
not clear. In the LIMITS study, an association between IL-6
and TNF-α and recurrent cerebrovascular events following
LS was demonstrated; however, sCD40 L and serum amyloid
A were not related to that risk.17 By contrast, various studies
suggested that sCD40 L and PF-4 were associated with risk
of vascular events, infarct size, and worse clinical courses
in stroke.18,19 Although the majority of patients in our cohort
that had radiological progression experienced LS or had
cognitive decline, we did not focus on clinical outcome in
the current analysis because this issue will be addressed in
Our findings supported a complex interaction between
pro-inflammatory mechanisms and platelet activation in SVD
that may result in ED and dysfunction of the neurovascular
unit.20 A number of experimental studies showed that white
matter is particularly vulnerable to inflammatory mediators
which contribute to the development of brain lesions and
neurological deficits in SVD.21 Increase in the permeability
of the blood–brain barrier (BBB) in SVD probably induces
a series of acute or chronic events leading to oxidative stress
and inflammatory mechanisms that promoting ED itself as
well as cumulative inflammatory cell infiltration resulting
in local or global brain damage.22 However, the cause of the
BBB dysfunction and inflammation – whether indicating
nonspecific responses to vascular risk factors, peripheral
systemic stimuli, systemic inflammation, or brain specific –
is not known.23 Diverse radiological features of SVD that
probably reflect different pathological mechanisms or disease
stages make the interpretation of experimental data
challenging. Lacunes are thought to represent the focal manifestation,
whereas WMLs denote a diffuse abnormality in the small
arterioles, resulting in a state of chronic hypoperfusion of
the white matter in the brain.23 Indeed, our study showed
diverse correlations between markers of radiological
progression and combined Z-scores for systemic and vascular
inflammation. Our data requires replication to ensure validity,
Currently, there is no blood biomarker that fulfils the
criteria of a surrogate endpoint in SVD; however, those
representing ED may prove useful in defining SVD heterogeneity,
especially at early stages of the disease, and, in combination
with neuroimaging markers, they may show better
correlation with clinical outcomes. Although the management of
traditional risk factors and antiplatelet treatment (for acute
LS) remain the most important preventive approach in SVD,
increasing evidence suggests that new studies should consider
drugs that target the endothelium and BBB to prevent and
There were several limitations to our study. The small
ww se number of patients may have produced biased estimates, and
:s na the results may not be generalizable to other populations and
should be considered exploratory in nature; however, this is
also a limiting factor in most published reports on this subject.
Multiple correlations could result in Type I error but
calculating the Z-score eliminates some issues raised by multiple
testing of single markers. Another weakness was the visual
assessment of the imaging; however, the SVD and Fazekas
scales remain the most widely used in the research literature.
This study was, therefore, regarded as hypothesis-generating
rather than definitive, and larger studies and replications
are needed for more robust conclusions. Although patients
with chronic inflammatory diseases were not included in the
study, blood biomarkers analyzed in the present study can
be either brain-specific or systemic markers. On the other
hand, our study had some advantages. We studied a
wellphenotyped group of patients with SVD that included VaP
and VaD subjects, and we analyzed a broad set of biomarkers
and their associations with the different imaging markers of
We showed that ED is not only a vascular disease marker
but plays a role in the radiological progression of SVD;
the concentrations of IL-6, PF-4, and sCD40 L were each
independently associated with an increased risk of any level
of radiological progression, as expressed by new lacunes or
WML progression. In addition, the two radiological subtypes
but, if validated, it lends support to the involvement of various
of SVD demonstrated differences in other associated
components of the inflammatory cascade in the pathogenesis
biochemical markers. Z-scores for systemic inflammation,
of WMLs and LIs. However, as SVD radiological markers
IL-6 and homocysteine levels predicted the development
frequently coexist, it is possible they share common
intrinof new lacunes, and the Z-scores for vascular inflammation
sic microvascular pathologies and, therefore, owing to its
and PF-4 were associated with the risk of WML
progresdiffuse nature, SVD should be regarded as a whole-brain
sion. Further research is required to ensure the validity of
The study was supported by the Polish Ministry of Science
and Higher Education as a research project (grant no N N402
The authors report no conflicts of interest in this work.
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