Temporal Profile of Molecular
Signatures Associated With Circulating
Endothelial Progenitor Cells in Human
Toma ´s Sobrino, Marı ´a Pe ´rez-Mato, David Brea, Manuel Rodrı ´guez-Ya ´n ˜ez,
Miguel Blanco, and Jose ´ Castillo*
Department of Neurology, Clinical Neurosciences Research Laboratory, Hospital Clı ´nico Universitario,
IDIS, University of Santiago de Compostela, Santiago de Compostela, Spain
Endothelial progenitor cells (EPC) have been associated
with good functional outcome in ischemic stroke. From
preclinical studies, it has been reported that EPC prolif-
eration is mediated by several molecular markers,
including vascular endothelial growth factor (VEGF),
stromal cell-derived factor-1a (SDF-1a), and the activity
of matrix metalloproteinase-9 (MMP-9). Therefore, our
aim was to study the role of these molecular factors in
EPC proliferation in human ischemic stroke. Forty-eight
patients with first episode of nonlacunar ischemic
stroke were prospectively included in the study within
12 hr of symptom onset. EPC colonies were classified
as early-outgrowth colony forming unit-endothelial cell
(CFU-EC) and quantified at admission, at 24 and 72 hr,
at day 7, and at 3 months. At the same time, serum
levels of VEGF, SDF-1a, and active MMP-9 were meas-
ured by ELISA. The primary endpoint was EPC incre-
ment during the first week, which was defined as the
difference in the number of CFU-EC between day 7
and admission. We found that VEGF (r 5 0.782), SDF-
1a (r 5 0.828), and active MMP-9 (r 5 0.740) levels at
24 hr from stroke onset showed a strong correlation
with EPC increment. Similar results were found for
VEGF levels at 72 hr (r 5 0.839) and at day 7 (r 5
0.602) as well as for active MMP-9 levels at 72 hr (r 5
0.442) and at day 7 (r 5 0.474). In the multivariate anal-
yses, serum levels of VEGF at 72 hr (B: 0.074, P <
0.0001) and SDF-1a at 24 hr (B: 0.049, P 5 0.008)
were independent factors for EPC increment during the
first week of evolution. These findings suggest that
VEGF and SDF-1a may mediate EPC proliferation in
human ischemic stroke.
C 2012 Wiley Periodicals, Inc.
Key words: adult
progenitor cells; molecular markers; outcome; cerebral
Circulating endothelial progenitor cells (EPC) have
been suggested to be a marker of cardiovascular risk and
endothelial function (Hill et al., 2003; Werner et al.,
2005). Moreover, EPC has been associated with good
neurological and functional outcome and reduced infarct
growth in patients after acute ischemic stroke (Sobrino
et al., 2007; Chu et al., 2008; Yip et al., 2008; Cesari
et al., 2009; Bogoslovsky et al., 2010). Likewise, experi-
mental and human studies indicate that EPC might
mediate endothelial cell regeneration and neovasculariza-
tion (Asahara et al., 1997; Werner et al., 2003; Kong
et al., 2004; Werner and Nickenig, 2006) and that EPC
participate in the cerebral neovascularization present in
adult brain after ischemia (Krupinski et al., 1994; Zhang
et al., 2000, 2002). These protective vascular effects
result from EPC proliferation.
On the other hand, several studies have shown that
the angiogenic factor vascular endothelial growth factor
(VEGF) promotes mobilization of EPC and incorpora-
tion of EPC into areas of neovascularization (Asahara
et al., 1999; Kalka et al., 2000; Hattori et al., 2001a). In
experimental models, administration of VEGF induces
an increase of EPC levels as well as an increased prolifer-
ative and migratory activity of EPC (Asahara et al.,
1999). In humans, exogenous administration of VEGF
Contract grant sponsor: Spanish Ministry of Science and Innovation;
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de Investigaciones Sanitarias, Instituto Salud Carlos III; Contract grant
number: RETICS-RD06/0026; Contract grant number: PI11/00909;
Contract grant sponsor: Xunta de Galicia (Consellerı ´a de Economı ´a e
Industria); Contract grant number: 10PXIB918282PR; Contract grant
sponsor: Xunta de Galicia (Consellerı ´a de Educacio ´n: Axudas para a
Consolidacio ´n e Estruturacio ´n de Unidades de Investigacio ´n Competiti-
vas do Sistema Universitario de Galicia); Contract grant number: N2011/
010; Contract grant sponsor: European Union program FEDER (Fondo
Europeo de Desarrollo Regional).
*Correspondence to: Prof. Jose ´ Castillo, Laboratorio de Investigacio ´n en
Neurociencias Clı ´nicas, Servicio de Neurologı ´a—Hospital Clı ´nico Uni-
versitario, c/Travesa da Choupana s/n, 15706 Santiago de Compostela,
Spain. E-mail: firstname.lastname@example.org
Received 20 October 2011; Revised 7 March 2012; Accepted 20 March
(wileyonlinelibrary.com). DOI: 10.1002/jnr.23068
online19 April2012 inWileyOnline Library
Journal of Neuroscience Research 90:1788–1793 (2012)
' 2012 Wiley Periodicals, Inc.
increases the number of circulating EPC (Kalka et al.,
2000), suggesting that its overexpression can induce the
mobilization of EPC. Moreover, other growth factors
such as angiopoietin-1 and fibroblast growth factor
(FGF) or chemokines such as stromal cell-derived factor
(SDF)-1a stimulate the mobilization and recruitment of
EPC (Hattori et al., 2001b; Yamaguchi et al., 2003).
Furthermore, the recruitment of progenitor cells from
the bone marrow seems to require the activity of matrix
metalloproteinase (MMP)-9. Knockout (KO) mice for
MMP-9 have a deficiency in the mobilization of stem
cells, and the administration of inhibitors of MMP
blocks the mobilization of EPC (Heissig et al., 2002).
Because EPC proliferation may be a therapeutic target in
cerebral ischemia, our aim was to study the role of these
molecular factors (VEGF, SDF-1a, and active MMP-9)
in EPC proliferation in human ischemic stroke.
MATERIALS AND METHODS
Study Population and Patients Characteristics
We studied 48 patients (male 50%, mean age 70.7 6
10.1 years) with a first-ever nonlacunar hemispheric cerebral
infarction in the territory of the middle cerebral artery of less
than 12 hr duration and previously independent for their daily
living activities. Exclusion criteria were chronic inflammatory
diseases, severe hepatic or renal diseases, hematological dis-
eases, cancer, infectious disease in the 15 days prior to inclu-
sion, no agreement to participate in the study, and lost to fol-
lowup. The ethics committee of Clinical University Hospital
of Santiago de Compostela approved the protocol, and
patients or their relatives gave informed consent.
All patients were admitted in the acute stroke unit and
treated by the same stroke team according to the Guidelines
of the Cerebrovascular Diseases Study Group of the Spanish
Society of Neurology (GEECV-SEN, 2004). Medical history
recording potential vascular risk factors, blood and coagulation
tests, 12-lead ECG, chest radiography, and carotid ultrasonog-
raphy were performed at admission. Stroke subtype was classi-
fied according to the TOAST criteria (Adams et al., 1993) as
atherothrombotic (n 5 8), cardioembolic (n 5 21), or unde-
termined (n 5 19). Fifteen patients received thrombolytic
treatment with recombinant tissue-plasminogen activator (rt-
PA) following the SITS-MOST criteria.
To evaluate neurologic deficit, the National Institutes of
Health Stroke Scale (NIHSS) was performed at admission; at
24, 48 6 6, and 72 6 24 hr; and at 7 6 1 and 90 6 7 days
(Brott et al., 1989). Functional outcome was evaluated at 3
months using the modified Rankin scale (mRS). Rankin
scores of 0, 1, and 2 indicate patients with independence in
their daily activities; 3, 4, and 5 indicate patients with depend-
ence for the daily activities; and a score of 6 identifies dead
patients. NIHSS and mRS were evaluated by internationally
certified neurologists. Good functional outcome was consid-
ered as an mRS ?2 at 3 months.
A multiparametric magnetic resonance imaging (MRI)
study at admission and at days 4–7 was performed in all
patients. Lesion volumes were calculated by using a manual
segmentation method: the perimeter of the area of abnormal
signal intensity was traced on each DWI or FLAIR map, and
subsequently the volumetric software estimated the total vol-
ume using the thickness and the traced area on each slice; the
window level and width were chosen to obtain the best con-
trast between the lesion and the normal surrounding tissue;
each volume calculation was performed three times, and the
mean value was taken as definitive. All neuroimaging evalua-
tions were made by the same neuroradiologist, who had no
knowledge of the patients’ clinical and laboratory results.
Isolation, Cultivation, and Characterization of EPC
We measured EPC according to methods described else-
where (Sobrino et al., 2007). The samples were processed
within 1 hr after collection by a unique investigator who had
no knowledge of the patients’ clinical and molecular results.
Briefly, a 14-ml sample of venous blood was used for the iso-
lation of mononuclear cells by Ficoll density gradient centrifu-
gation. Five million peripheral blood mononuclear cells per
well were then plated on fibronectin-coated six-well dishes
(BD BioSciences Discovery Labware) in Endocult Liquid Me-
dium (Stem Cell Technologies) for 2 days. After that, one
million nonadherent cells were harvested and plated on fibro-
nectin-coated 24-well plates (BD BioSciences Discovery Lab-
ware). Colonies formed 3 days later were counted and classi-
fied as early-outgrowth colony forming unit-endothelial cell
(CFU-EC). CFU-EC quantification, in a minimum of three
wells per sample, was performed at admission, at day 7 6 1,
and at 3 months 6 7 days from stroke onset. We calculated
the increment of EPC colonies during the first week as the
absolute difference between the number of CFU-EC at day 7
and admission. The intraobserver correlation was 0.96.
Blood samples, drawn from all patients at admission, at
24 6 6 and 72 6 24 hr, at day 7 6 1, and at 3 months 6 7
days were collected in glass test tubes, centrifuged at 3,000g
for 10 min, and immediately frozen and stored at 2808C. Se-
rum VEGF and SDF-1a levels were measured with commer-
cially available quantitative enzyme-linked immunosorbent
assay (ELISA) kits obtained from Bender MedSystems and
RayBiotech Inc., respectively. Finally, endogenous serum lev-
els of active MMP-9 were measured with an Activity Assay
System from Biotrak, Amersham Biosciences. Determinations
were performed in an independent laboratory blinded to clini-
cal and neuroimaging data.
The results are expressed as percentages for categorical
variables and as mean (SD) or median [quartiles] for the con-
tinuous variables depending on normal distribution or not.
Pearson’s analyses were used for bivariate correlations. A re-
ceiver operator characteristic (ROC) analysis was conducted
to determine the best cutoff value of the EPC colony incre-
Molecular Markers Associated With EPC in Stroke 1789
Journal of Neuroscience Research
ment during the first week for the prediction of good out-
come (mRS ?2 at 3 months).
Because of the colinearity between the molecular
markers, the influence of each them on EPC increment dur-
ing the first week was assessed in a separate multiple linear
regression models. This association will be considered inde-
pendent when molecular markers determine the value of EPC
increment during the first week, not depending on other vari-
ables, which may be influencing the main dependent variable
(EPC increment during the first week). Results were
expressed as B with the corresponding 95% confidence inter-
vals (95% CI). P < 0.05 was considered significant in all tests.
The statistical analysis was conducted in SPSS 16.0 (SPSS,
Chicago, IL) software.
We prospectively studied 48 nonlacunar ischemic
stroke patients (male 50%; mean age 70.7 6 10.1 years).
Median [quartiles] NIHSS score on admission was 12 [6,
18], and mean time from stroke onset was 5.6 [2.4, 8.5]
hr. DWI ischemic lesion at admission was 24.6 6
Temporal Profile of Molecular Markers
Figure 1 shows the temporal profile of molecular
markers during the first 3 months from stroke onset.
The highest peak value was observed at 24 hr for SDF-
1a and active MMP-9 and on day 7 for VEGF,
although the largest increase occurs in the first 24 hr for
all molecular markers. Serum levels of all markers,
except for VEGF, decrease sharply after 72 hr. For
VEGF levels, this decline occurred from day 7. More-
over, the number of CFU-EC increased progressively
during the first 3 months from stroke onset (Fig. 2; all
P < 0.0001).
Relationship of CFU-EC Counts With NIHSS
Scores and DWI Lesion Volumes
CFU-EC numbers at admission were not associated
with baseline NIHSS (r 5 20.140, P 5 0.344), NIHSS
at day 7 (r 5 20.261, P 5 0.067), or NIHSS at 3
months (r 5 20.248, P 5 0.089). However, CFU-EC
increment during the first week negatively correlated
with NIHSS at day 7 (r 5 20.696, P < 0.0001) and at
3 months (r 5 20.696, P < 0.0001). Similarly, we
found no correlations between CFU-EC counts at
admission and DWI ischemic lesions at baseline (r 5
20.186, P 5 0.206) or at days 4–7 (r 5 20.066, P 5
0.654). Nevertheless, CFU-EC increment during the
first week was negatively associated with DWI ischemic
lesion at days 4–7 (r 5 20.496, P < 0.0001).
Temporal Profile of Molecular Signatures
Associated With CFU-EC Levels
No correlations were found between serum levels
of molecular markers at admission and any measure of
EPC levels (CFU-EC number at admission, CFU-EC
Fig. 1. Temporal profile of serum levels of VEGF, SDF-1a, and
active MMP-9 during the first 3 months from stroke onset.
1790 Sobrino et al.
Journal of Neuroscience Research
increment during the first week, and CFU-EC number
at 3 months). However, we found that VEGF (r 5
0.782), SDF-1a (r 5 0.828), and active MMP-9 (r 5
0.740) serum levels at 24 hr from stroke onset showed a
strong correlation with CFU-EC increment during the
first week (all P < 0.0001). Similar results were found
for VEGF levels at 72 hr (r 5 0.839, P < 0.0001) and
at day 7 (r 5 0.602, P < 0.0001) as well as for active
MMP-9 levels at 72 hr (r 5 0.442, P 5 0.002) and at
day 7 (r 5 0.474, P 5 0.001). On the other hand,
CFU-EC number at 3 months was correlated with se-
rum levels of VEGF at 24 hr (r 5 0.717), 72 hr (r 5
0.727), and day 7 (r 5 0.669; all P < 0.0001); SDF-1a
at 24 hr (r 5 0.587, P < 0.0001); and active MMP-9 at
24 hr (r 5 0.561, P < 0.0001), 72 hr (r 5 0.343, P 5
0.024), and day 7 (r 5 0.403, P 5 0.007). Finally, se-
rum levels of VEGF, SDF-1a, and active MMP-9 at 3
months did not correlate with the number of CFU-EC
at 3 months.
Primary Endpoint: EPC Increment During the
According to the ROC analysis, an EPC increment
?4 CFU-EC predicted with the highest sensitivity
(88%) and specificity (92%) the probability of good func-
tional outcome at 3 months (area under the curve 0.903,
95%CI 0.811–0.995, P < 0.0001). For that reason, our
multivariate analysis was focused on those molecular
markers, which were positively associated with the EPC
increment during the first week.
As mentioned above, CFU-EC increment during
the first week was correlated with serum levels of VEGF
and active MMP-9 at 24 hr, 72 hr, and day 7. Likewise,
a strong correlation was found between CFU-EC incre-
ment during the first week and SDF-1a levels at 24 hr.
In the multivariate analyses, serum levels of VEGF
at 72 hr (B: 0.074, IC 95% 0.058–0.090, P < 0.0001)
and SDF-1a at 24 hr (B: 0.049, IC 95% 0.014–0.084, P
5 0.008) were the only independent factors for EPC in-
crement during the first week (Table I).
This prospective study has evaluated the temporal
profile of molecular signatures associated with circulating
EPC in human ischemic stroke. Remarkably, EPC in-
crement during the first week was correlated with serum
levels of VEGF and active MMP-9 at 24 hr, 72 hr, and
day 7 as well as with SDF-1a levels at 24 hr. However,
the serum levels of VEGF at 72 hr and SDF-1a at 24 hr
were the only independent factors for EPC increment
during the first week.
We have demonstrated that, in human ischemic
stroke, serum levels of VEGF, SDF-1a, and active
MMP-9 increase in response to cerebral ischemia within
the first 24 hr from symptom onset and that the magni-
tude of this increase is directly related to an EPC incre-
ment during the first week. These findings are in line
with other clinical studies demonstrating increased levels
of these molecular markers during the acute phase of is-
chemic stroke (Bogoslovsky et al., 2011a,b). Likewise,
Bogoslovsky et al. found a strong correlation between
serum levels of SDF-1a and EPC count, but not for
VEGF and MMP-9. This controversy may be due to the
temporal profiles studied for molecular markers, and
EPC were different in both studies, insofar as molecular
marker levels and EPC counts were measured only at
days 1 and 3 from stroke onset in the studies of Bogo-
slovsky et al. Furthermore, we measured the endogenous
serum levels of active MMP-9, whereas total levels of
MMP-9 were determined in the study of Bogoslovsky
et al. (2011a,b). On the other hand, the fact that serum
levels of molecular markers at 24 hr correlated with
EPC increment during the first week, but not at admis-
sion, and that EPC increment during the first week, but
TABLE I. B of CFU-EC Increment During the First Week for
B 95% CIP
SDF-1a levels at 24 hr
Active MMP-9 levels
*Note that, because of the colinearity between the molecular markers,
the influence of each them on EPC increment during the first week was
assessed in a separate multiple linear regression model. VEGF, vascular
endothelial growth factor; SDF-1a, stromal cell-derived factor-1a;
MMP-9, matrix metalloproteinase 9.
Fig. 2. Temporal profile of the number of early-outgrowth colony
forming unit-endothelial cell (CFU-EC) during the first 3 months
from stroke onset.
Molecular Markers Associated With EPC in Stroke1791
Journal of Neuroscience Research
not CFU-EC counts at baseline, has been associated
with better neurological outcome and reduced infarct
growth supports the hypothesis that cerebral ischemia
induces the activation of molecular pathways of EPC
mobilization focused on promoting endogenous proc-
esses of vascular and neurorepair.
Several studies have demonstrated that VEGF pro-
motes the mobilization and incorporation of EPCs to
the neovascularization areas (Asahara et al., 1999; Kalka
et al., 2000; Hattori et al., 2001a). In addition, the
increase of circulating EPC has been correlated with
VEGF serum levels, which also peak at day 7, in patients
with acute myocardial infarction (Shintani et al., 2001).
On the other hand, the activity of MMP-9 causes a mas-
sive release of stem cell factor (SCF), which favors the
recruitment of progenitor cells, among which are EPC,
from bone marrow. Moreover, the mechanism by which
placental growth factor (PGF) recruits progenitor cells
derived from bone marrow requires the activity of
MMP-9, which induces the release of SCF (Hattori
et al., 2002). Likewise, active MMP-9 induces the
release of cytokines that cause the mobilization of quies-
cent EPC (Rafii et al., 2002). Finally, our results would
come to support the studies suggesting that SDF-1a
could contribute to the homing of EPC to areas of neo-
vascularization and re-endothelialization (Aiuti et al.,
1997; Salcedo et al., 1999; Hattori et al., 2001; Yamagu-
chi et al., 2003). However, these molecular mechanisms
may be interrelated, in that our results and those
obtained in other studies have demonstrated that MMP-
9 plays an important role in progenitor cells mobilization
from bone marrow and focal angiogenesis in the brain in
response to VEGF stimulation (Heissig et al., 2002; Lee
et al., 2009; Hao et al., 2011). Likewise, SDF-1a indu-
ces the expression of VEGF and mediates angiogenesis
in vivo (Salcedo et al., 1999).
On the other hand, serum levels of VEGF and
SDF-1a increased progressively during the first 3 days
following ischemic stroke. Previous studies by our group
have demonstrated similar results in patients with intra-
cerebral hemorrhage (ICH; Sobrino et al., 2009, 2011).
We also found in ICH patients a strong correlation
between VEGF and SDF-1a serum levels and circulating
concentrations of bone marrow-derived progenitor cells
(BMPCs) at day 7 (Sobrino et al., 2011). Given that the
EPC is a subtype of BMPCs, it is tempting to postulate
that similar molecular and cellular mechanisms are
involved in the two major subtypes of stroke (ischemic
and hemorrhagic stroke).
This study has a number of limitations. First, this is
a secondary analysis of a prospective study whose main
objective was to analyze the prognostic value of EPC in
functional outcome in patients with acute ischemic
stroke. Second, we did not determine the levels of EPC
or molecular markers at longer intervals from day 7 to 3
months of evolution, so we cannot conclude whether
EPC number at 3 months was associated with an acute
and transient increase in molecular levels or was the
result of a sustained increase of these molecular markers
during the followup. There are different methods for the
study of EPC, such as flow cytometry or cell culture for
CFU-EC counts. Currently, the most well accepted is
flow cytometry (Yoder et al., 2007; Rouhl et al., 2008),
but several studies in stroke patients have shown virtually
the same results using both techniques (Sobrino et al.,
2007; Chu et al., 2008; Yip et al., 2008). However, the
robustness of the results supports the need to explore the
role of these molecular markers, especially VEGF and
SDF-1a, as a new therapeutic tool able to increase cir-
culating EPC levels after ischemic stroke.
In conclusion, VEGF and SDF-1a may mediate
EPC proliferation and vascular repair after acute ische-
mic stroke. The role of these molecules as a new thera-
peutic tool able to promote endogenous neurorepair of
brain tissue damaged by an increase of EPC should be
The funders had no role in study design, data col-
lection and analysis, decision to publish, or preparation
of the manuscript.
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