morbidity.1,2 Whereas the burden of cerebrovascular disease
may be largely attributed to well-established stroke risk factors,
genetic and lifestyle differences and structural and functional
brain capacities (such as neural density, synaptic connectivity,
and the compensatory ability of existing neural networks) may
modulate risk and response to vascular brain injury.3,4 Therefore,
erebrovascular disease (overt and covert) accounts for a
significant proportion of brain aging–related mortality and
searching for novel pathways involved in cerebrovascular dis-
ease may improve our understanding of disease mechanisms
and help facilitate discovery of new molecular targets for stroke
therapy and prevention. Brain-derived neurotrophic factor
(BDNF) and vascular endothelial growth factor (VEGF), both
present during early neuronal development, have also been
found to have a role in maintaining neuronal plasticity and in
neuronal repair by promoting neurogenesis and angiogenesis.5,6
Background and Purpose—Brain-derived neurotrophic factor (BDNF), a major neurotrophin and vascular endothelial
growth factor (VEGF) have a documented role in neurogenesis, angiogenesis, and neuronal survival. In animal
experiments, they impact infarct size and functional motor recovery after an ischemic brain lesion. We sought to
examine the association of serum BDNF and VEGF with the risk of clinical stroke or subclinical vascular brain injury
in a community-based sample.
Methods—In 3440 Framingham Study participants (mean age, 65±11 years; 56% women) who were free of stroke/transient
ischemic attack (TIA), we related baseline BDNF and logVEGF to risk of incident stroke/TIA. In a subsample with brain
MRI and with neuropsychological tests available (n=1863 and 2104, respectively; mean age, 61±9 years, 55% women,
in each), we related baseline BDNF and logVEGF to log-white matter hyperintensity volume on brain MRI, and to
visuospatial memory and executive function tests.
Results—During a median follow-up of 10 years, 193 participants experienced incident stroke/TIA. In multivariable analyses
adjusted for age, sex, and traditional stroke risk factors, lower BDNF and higher logVEGF levels were associated with an
increased risk of incident stroke/TIA (hazard ratio comparing BDNF Q1 versus Q2–Q4, 1.47; 95% confidence interval,
1.09–2.00; P=0.012 and hazard ratio/SD increase in logVEGF, 1.21; 95% confidence interval, 1.04–1.40; P=0.012).
Persons with higher BDNF levels had less log-white matter hyperintensity volume (β±SE=−0.05±0.02; P=0.025), and
better visual memory (β±SE=0.18±0.07; P=0.005).
Conclusions—Lower serum BDNF and higher VEGF concentrations were associated with increased risk of incident stroke/
TIA. Higher levels of BDNF were also associated with less white matter hyperintensity and better visual memory.
Our findings suggest that circulating BDNF and VEGF levels modify risk of clinical and subclinical vascular brain
injury. (Stroke. 2013;44:2768-2775.)
Key Words: brain-derived neurotrophic factor ◼ brain MRI ◼ risk ◼ stroke ◼ subclinical
◼ vascular endothelial growth factor A
Serum Brain–Derived Neurotrophic Factor and Vascular
Endothelial Growth Factor Levels Are Associated With Risk
of Stroke and Vascular Brain Injury
Aleksandra Pikula, MD; Alexa S. Beiser, PhD; Tai C. Chen, PhD;
Sarah R. Preis, ScD, MPH; Demetrios Vorgias, MSc; Charles DeCarli, MD; Rhoda Au, PhD;
Margaret Kelly-Hayes, EdD, RN; Carlos S. Kase, MD; Philip A. Wolf, MD;
Ramachandran S. Vasan, MD; Sudha Seshadri, MD
Received March 12, 2013; accepted July 8, 2013.
From the Boston University Schools of Medicine and Public Health, Boston, MA (A.P., A.S.B., T.C.C., S.R.P., D.V., R.A., M.K.-H., C.S.K., P.A.W.,
R.S.V., S.S.); Framingham Heart Study, Framingham, MA (A.P., A.S.B., S.R.P., C.D., R.A., M.K.-H., C.S.K., P.A.W., R.S.V., S.S.); and University of
California at Davis, Sacramento (C.D.).
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.
Correspondence to Aleksandra Pikula, MD, Department of Neurology, Boston University School of Medicine, 715 Albany St, D-312, Boston,
MA 02118-2526. E-mail email@example.com
© 2013 American Heart Association, Inc.
Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.113.001447
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Pikula et al BDNF, VEGF, and Risk of Stroke, White Matter 2769
Low circulating BDNF concentrations have been observed
in patients with coronary artery disease, type 2 diabetes melli-
tus, metabolic syndrome, acute coronary syndrome,7 and phys-
ical inactivity,8 and higher VEGF concentrations in patients
with hypertension, type 2 diabetes mellitus, smoking, obesity,
and coronary artery disease,9,10 all risk factors associated with
stroke.11 Interestingly, under hypoxic and ischemic condi-
tions, both BDNF and VEGF and their respective receptors are
upregulated in neurons and vascular endothelial cells.12,13
To our knowledge, to date there has been no prospective
or clinical study that has systematically examined the role of
BDNF and VEGF in determining the risk of clinical stroke or
subclinical vascular brain injury in a community-based popu-
lation. We proposed to examine an association between serum
BDNF and VEGF levels and (1) risk of stroke/transient isch-
emic attack (TIA), (2) brain MRI measures of subclinical vas-
cular injury (white matter hyperintensity volume [WMHV]),
and (3) performance on executive function and visual memory
tasks; the cognitive domains previously associated with vas-
cular risk factors and WMHV.14
The Framingham Study is a prospective, community-based cohort
that enrolled 5209 participants in 1948; these participants have had
biennial re-examinations.15 In 1971, the offspring of the Original co-
hort and their spouses were enrolled in an Offspring cohort, assessed
once every 4 years.16 The participants from both cohorts have been
under ongoing surveillance for incident stroke and TIA since the in-
ception of the study.
Stroke- and TIA-free participants in the Original cohort who at-
tended examination 23 (n=581; 80±4 years; 64% women), and
Offspring participants who attended examination 7 (n=2859; 62±9
years; 54% women) were included if they also had available BDNF
and VEGF levels with no missing covariate data (Study Sample 1;
n=3440); only 88 persons (3%) were excluded from Study Sample 1
for missing covariate data.
Offspring participants who attended examination 7 were also
invited to undergo cognitive testing and brain MRI (1999–2005).
Among the 3020 persons who attended examination 7 and had avail-
able BDNF and VEGF levels, 68 persons were excluded for miss-
ing covariate data. Of the remaining 2952 persons, 2186 persons had
a neuropsychological (NP) battery administered, and 1945 persons
underwent brain MRI; 82 persons were excluded for having stroke,
dementia, or other neurological illness (such as multiple sclerosis,
severe head trauma, or brain tumor) that could affect MRI/NP mea-
sures; we did not exclude persons with TIA in this analysis. The rest
were eligible for investigation of the association between BDNF and
VEGF and (1) WMHV and (2) executive function and visual mem-
ory (Study Samples 2a: n=1863; 61±9 years; 55% women and 2b:
n=2104; 61±9 years; 55% women).
Laboratory Measurements of BDNF and VEGF
Serum BDNF and VEGF concentrations were measured using com-
mercial ELISA assays obtained from R&D Systems (Minneapolis,
MN) on previously frozen (stored at −70ºC) blood samples drawn in
the fasting state from persons who attended the 23rd Original cohort
and the 7th Offspring examinations. The intra-assay and interassay
coefficients of variation for BDNF and VEGF were 4.8 and 7.6%, and
for VEGF 3.9 and 8.4%, respectively.
The age and sex-adjusted Pearson correlation coefficient between
BDNF and logVEGF was 0.15 (P<0.001) for the Original cohort,
0.23 (P<0.001) for the Offspring cohort, and 0.22 (P<0.001) for both
Stroke/TIA (Clinical Outcome)
Our protocols for stroke surveillance and for establishing the diag-
nosis and type of stroke have been previously published.11 Stroke
was defined as an acute onset focal neurological deficit of presumed
or definite vascular pathogenesis, persisting for >24 hours and TIA
symptoms lasting for ≤24 hours. When available we also used imag-
ing studies and other laboratory criteria, noninvasive vascular stud-
ies, cardiac evaluations for a source of embolus, and also information
from autopsy studies; these data were also used to categorize strokes
as ischemic or hemorrhagic.
Brain Imaging Measures (Subclinical Outcome)
MRI acquisitions and measurement techniques and inter-rater reliabil-
ity scores have been previously published.17 In summary, digital MRI
information once acquired on a 1 or 1.5 Tesla Siemens Magnetom
and transferred to the centralized imaging center at the University of
California-Davis Medical center was analyzed by operators blinded
to the participant’s demographics, exposure to stroke risk factors,
and BDNF and VEGF levels. The volume of abnormal WMH was
determined by using previously described semiautomated methods,
adjusted for total cranial volume and log-transformed (log-WMHV).
Participants whose log-WMHV was >1 SD above the age-specific
mean were categorized as having extensive WMHV (ext-WMHV).
Subjects were administered (typically on the same day as their MRI)
a half-hour NP battery using standard administration protocols and
trained examiners. Details of the tests administered and normative
values for the Framingham Original and Offspring cohorts have
been previously published.18,19 In this study, we selected cognitive
domains previously associated with vascular risk factors and vascu-
lar brain injury; visuospatial memory (measured by delayed recall
on the visual reproduction test [VR-d] from the Wechsler Memory
Scale) and executive function (measured by Trail Making Test B-A).
We transformed Trails B-A using a log-transformation to normalize
its distribution and changed its sign, so that smaller numbers indicate
Definition of Covariates
We used previously described and validated components of the
Framingham Stroke Risk Profile (FSRP),11 as baseline covariates.
Educational achievement was defined as no high-school degree,
high-school degree only, or more than high-school degree (college
degree). Depressive symptoms were evaluated with the Center for
Epidemiological Studies Depression scale (CES-D).20 The physical
activity index was calculated as a composite score based on infor-
mation collected from a structured questionnaire.21 Both CES-D and
physical activity index were log-transformed for analysis.
BDNF and VEGF were analyzed as continuous variables. VEGF
values were log-transformed to normalize the skewed distribution of
VEGF. We used Cox-proportional hazards models to relate baseline
serum BDNF and logVEGF levels (separately) to risk of incident
stroke/TIA in Study Sample 1. We first examined the linear effect of a
1-SD unit increase/decrease in BDNF and logVEGF on risk of stroke/
TIA. We also explored threshold effects comparing the risk across
quartiles of BDNF and VEGF. We observed a threshold effect with
BDNF only. Therefore, we ran threshold models for BDNF using the
lowest quartile, Q1, as the reference and also assessed a threshold
model comparing risk for persons in Q1 with persons in Q2 to Q4.
We performed a similar set of analyses on a Study Sample 1b with
stroke alone as the outcome. Using multivariable linear and logis-
tic regression models, we examined the cross-sectional associations
between BDNF and logVEGF and markers of brain aging (WMHV,
by guest on October 30, 2015http://stroke.ahajournals.org/Downloaded from
Supplementary Table V. Additional effect of serum BDNF and VEGF on risk of stroke/TIA
†Model B HR [95% CI] P NRI [95%CI] C-Statistics
Q1 vs Q2-
Model with both markers included
Q1 vs Q2-
†Model B: Model A additionally adjusted for SBP, CVD, DM, AF and SMK
Note: the interaction between log(VEGF) and Q1-BDNF has p=0.465.
Vasan and Sudha Seshadri
DeCarli, Rhoda Au, Margaret Kelly-Hayes, Carlos S. Kase, Philip A. Wolf, Ramachandran S.
Aleksandra Pikula, Alexa S. Beiser, Tai C. Chen, Sarah R. Preis, Demetrios Vorgias, Charles
Levels Are Associated With Risk of Stroke and Vascular Brain Injury: Framingham
Derived Neurotrophic Factor and Vascular Endothelial Growth Factor
Print ISSN: 0039-2499. Online ISSN: 1524-4628
Copyright © 2013 American Heart Association, Inc. All rights reserved.
is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
2013;44:2768-2775; originally published online August 8, 2013;
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