European Journal of Pharmacology 711 (2013) 27–35

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European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Cardiovascular pharmacology

Irbesartan administration therapeutically influences circulating

endothelial progenitor cell and microparticle mobilization by
involvement of pro-inflammatory cytokines
Adriana Georgescu a,b,n,1, Nicoleta Alexandru a,b,n,1, Miruna Nemecz b,
Irina Titorencu a,b, Doina Popov b
a
Petru Poni’ Institute of Macromolecular Chemistry, Iasi, Romania
b
Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of Romanian Academy, Bucharest, Romania

art ic l e i nf o a b s t r a c t

Article history: Circulating microparticles (MPs) and endothelial progenitor cells (EPCs) correlate with endothelial
Received 3 December 2012 dysfunction and contribute to the pathogenesis of atherosclerosis. In this context, we explored whether
Received in revised form the angiotensin II type I receptor antagonist, irbesartan, exerts a pharmacological control in the
27 March 2013
atherosclerotic process by the improvement of EPC mobilization and inhibitory effects on MP release
Accepted 3 April 2013
Available online 29 April 2013
and VEGF and SDF-1α levels in the hypertensive–hypercholesterolemic (HH) hamster model. The HH
hamsters were treated with irbesartan (50 mg/kg b.w/day administered by gavage) for 4 month (HHI).
Keywords: We analyzed MP/EPC infiltration in vascular wall before and after irbesartan administration as well as the
Irbesartan endothelial function and expression of VEGF/SDF-1α in plasma and tissue and of molecular pathways
Endothelial progenitor cells
activated by them. The results showed that treatment with irbesartan significantly increased EPC
Microparticles
infiltration and decreased MP infiltration. The mechanisms underlying this response include the
Atherosclerosis
reduction/increase of a number of specific membrane receptors exposed by MPs (TF, P-Selectin,
E-Selectin, PSGL-1, Rantes), respectively, by EPCs (β2-Integrins, α4β1-integrin), the augmentation of
endothelium-mediated vasodilation and the reduction of protein expression of VEGF/SDF-1α followed
by: (1) the diminishment of pro-inflammatory endothelial cytokines: VEGFR1, VEGFR2, CXCR4, Tie2, PIGF
with role in EPC homing to sites of damaged endothelium; and (2) the increase of protein expression of
COX-2, PGI2 synthase molecules with role in the improvement of arterial wall vasodilatation. In
conclusion, the study underlines that irbesartan administration therapeutically improves/reduces EPC,
respectively, MP mobilization and this action may be of salutary relevance contributing to its beneficial
cardiovascular effects.
& 2013 Elsevier B.V. All rights reserved.

1. Introduction ratio of circulating MPs to endothelial progenitor cells (EPCs) may

Atherosclerosis is one of the most important and common
cause of death and disability in the world and remains an occult
but important precursor of significant cardiovascular events.
It is now increasingly admitted that cell-derived microparticles
(MPs) may contribute to the initiation, progression and clinical
complications of cardiovascular diseases, and appear as interesting
biomarkers to predict this pathology (Baron et al., 2011). Also, the
n
Corresponding authors at: Pathophysiology and Pharmacology Department,
Institute of Cellular Biology and Pathology ‘Nicolae Simionescu’ of Romanian
Academy, 8, BP Hasdeu Street, P.O. Box 35-14, 050568-Bucharest, Romania.
Tel.: +4021 319 4518; fax: +4021 319 4519.
E-mail addresses: adriana.georgescu@icbp.ro (A. Georgescu),
nicoleta.alexandru@icbp.ro (N. Alexandru).
1
www.icbp.ro.
be a new and valuable cellular marker of vascular dysfunction in
atherosclerosis (Georgescu et al., 2012). Several reports emphasize
the contribution of EPCs to new vessel formation in different
physiological and pathological settings. Vascular endothelial
growth factor (VEGF) and its receptor (VEGFR) are crucial regula-
tors of the growth, development, and differentiation of heart and
blood vessels (Wagner and Siddiqui, 2007). The therapeutic effect
of VEGF consists in the improvement of heart structure and
function (Yockman et al., 2009; Hao et al., 2007). VEGF was
described as an effective mobilizer of EPCs and a potent inducer
of angiogenesis (Zampetaki et al., 2008). Also, the chemokine SDF-
1α (stromal cell derived factor-1α) and its receptor CXCR4 (CXC
chemokine receptor 4) play a major role in the recruitment and
retention of stem cells to ischemic areas (Askari et al., 2003;
Lapidot et al., 2005). Concerning integrins, very little is known in
relation to EPCs. In human adult peripheral blood-derived EPCs,

0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.04.004
28 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35
Fig. 1. Chemical formula and structure of irbesartan.
transendothelial migration appears to be mediated mainly by β2
integrins and depends on MCP-1 (Monocyte chemoattractant
protein-1) and VEGF (Chavakis et al., 2007). This is further
supported by a study showing that increased expression of
ICAM-1 (Intercellular adhesion molecule-1) in endothelial cells,
via Akt (Protein kinase B or PKB) activation, increases EPC homing
and transendothelial migration in vitro assays (Hur et al., 2007).
On the other hand, there are a lot of pharmacological approa-
ches on mobilization and functional activity of EPCs to improve the
endothelial repair mechanisms. In particular, several experimen-
tal and clinical studies have suggested that statins, angiotensin-
converting enzyme inhibitors, angiotensin II type 1 receptor blockers,
PPAR-γ (Peroxisome proliferator-activated receptor γ) agonists and
erythropoietin increase the number and functional activity of EPCs
(Besler et al., 2008). Initial evidence that angiotensin II type 1 receptor
antagonists affect the number of circulating EPCs was obtained in
patients with Type 2 diabetes treated with olmesartan or irbesartan
for 12 weeks (Bahlmann et al., 2005). Treatment with these antago-
nists increases the EPC number in peripheral blood, with a significant
effect in irebesartan group already apparent after 4 weeks of therapy.
Studies on hypertensive–hypercoleterolemic hamsters have shown
that irbesartan augments the numbers of EPC in peripheral blood and
exerts a favorable effect on atherosclerotic plaque formation at least in
part by inhibiting specific proinflammatory molecules (Georgescu
et al., 2012). The underlying mechanisms remain largely to be defined.
The prevention and treatment atherosclerosis-related macro-
and microvascular complications dramatically influence the life
expectance and the quality of life of atherosclerotic patients, thus
representing a crucial target for physicians. Irbesartan (Fig. 1)
therapy might represent potential future strategy to mobilize EPCs,
finally leading to a better cardiovascular outcome.
2. Material and methods
2.1. Ethics statement
All the protocols were approved by the Ethics Committee of
ICBP ‘N. Simionescu’ (Permit Number: 421), and were conducted in
accordance with the Guide for the Care and Use of Laboratory
Animals published by the US National Institutes of Health (NIH
publication no 85-23, revised 1996). Also, the research on these
animals was performed according to procedures defined in the
Council Directive 86/609/EEC of 24 November 1986.
2.2. Animal models
180 male Golden Syrian hamsters (3 months old) were divided
into three equal experimental groups:(i) simultaneously hyperten-
sive–hyperlipidemic (HH) by combining two feeding conditions:
the standard chow supplemented with 3% cholesterol and 15%
butter, for hyperlipemia, and 8% NaCl diet, for hypertension, for
4 months (Calhoun et al., 1995), (ii) simultaneously hypertensive–
hyperlipidemic treated with irbesartan (50 mg/kg b.w/day admi-
nistered by gavage for 4 months) (HHI) (Riveiro et al., 2002) and
(iii) controls (C), age-matched normal healthy animals which were
kept in the same housing conditions and fed a standard chow
containing basal 1% NaCl. At 16 weeks after the beginning of
experiment, the hamsters were sacrificed for biochemical, struc-
tural and functional assays.
2.3. Characterization of experimental models
After 4 months of diet, the systolic and diastolic arterial blood
pressure and the heart rate were recorded in the presence of
10 U/ml heparin using a Physiological Pressure Transducer (model
MLT844/D) connected to a PowerLab data acquisition unit (ADIn-
struments, Sydney, Australia), for all groups of animals. Arterial
blood pressure and heart rate were measured invasively by placing
a cannula into abdominal artery and connecting it to an electronic
pressure transducer (Georgescu et al., 2007).
The plasma glucose, cholesterol and triglyceride concentrations
were assayed at the end of the diet period of 4 months, and the
monitorization of values was made using the Accutrend GCT
(Roche, USA) apparatus. Also, the blood was collected on 0.138 M
tri–sodium citrate 9/1 (vol/vol) and thoracic aortic arch and
mesenteric resistance arteries were explanted from every animal
considered in this study.
2.4. Immunohistochemistry detection of MPs and EPCs
in vascular wall
It was explored the adhesion and homing capacity of MPs and
EPCs to arterial wall by immunofluorescence microscopy. The thin
sections of tissue (7 mm) were deparaffinized, blocked with 3% BSA in
PBS, washed with PBS, incubated with 0.1% NaBH4 in PBS, washed
with PBS, incubated with Annexin V-FITC (Annexin V (FL): sc-4252,
Santa Cruz Biotechnology), for MPs, or CD34-PE (CD34 Antibody
(ICO115): sc-7324, Santa Cruz Biotechnology), for EPC, for 2 h at room
temperature in dark, washed with 1% BSA in PBS, washed again in
PBS, mounted in 90% glycerol and observed under fluorescence
microscope For image analysis, NIS-Elements BR (Nikon) software
was employed.
2.5. Preparation of platelet free plasma, the source for
circulating MPs
Plasma MPs were separated according to the method reported
by Georgescu et al. 2012. Briefly, the procedure consisted in
collection of blood, centrifugation at 1000g for 15 min at 15 1C,
and separation of platelet rich plasma (PRP). The latter was further
centrifuged at 2500g for 15 min at 15 1C, and the platelet free
plasma (PFP) was obtained. Centrifugation of PFP at 13,000g for
5 min at 15 1C allowed collection of MPs in supernatant.
2.5.1. Sorting MPs by flow cytometry
Total MPs from plasma were characterized using specific anti-
body Annexin V FITC (for phosphatidylserine, PS). The reaction
consisted in: 20 μl PFP and 10 μl Annexin V-FITC, mixing and
incubation in the dark for 40 min at room temperature. Freshly
filtered PBS buffer (200 μl) was added right before detection.
Cell sorting involves physical separation of MPs from suspen-
sion stained with specific fluorescent dye (as above). MPs were
gated as events with a 0.1 μm to 1.0 μm diameter identified in
forward-scatter and side-scatter intensity dot-plot representa-
tion using standards synthetic beads of 1 μm in diameter. MPs
 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35
concentration was assessed by comparison with calibrator Flow-
count beads (10 μm in diameter) with a predetermined concentra-
tion. The experiments were conducted using the Flow cytometer
MoFlo (Dako, USA).
2.6. Functional investigation of the arterial wall
2.6.1. Mesenteric arterial bed preparation
Animals were sacrificed by cervical dislocation and the small
intestine was excised. The isolated arterial segments (third order)
were mounted as a ring preparation in the wire-myograph (Model
410A, J.P. Trading, Denmark) as described by Mulvany and Halpern
(1977).
2.6.2. The myograph technique
The myograph chamber was filled with HEPES salt solution
maintained at 37 1C and continuously gassed with O2. After an
equilibration period of 20 min, the internal circumference of
mounted artery was normalized on the basis of passive tension–
length curve to a value that gives maximal force development.
Then, for each vessel a routine “run-up” procedure consisting
of consecutive contractions to 5
10−6 M noradrenaline (NA),
125
10−3 M potassium chloride (KCl), and 5
10−6 M noradrena-
line in 125
10−3 M potassium chloride was performed.
To measure the acetylcholine (ACh)—induced relaxation, the
vessels were first contracted in noradrenaline (10−8–10−4 M), and
then exposed at 2 min intervals to increasing concentrations of
acetylcholine (10−8–10−4 M).
2.7. Enzyme-linked immunosorbent assay (ELISA)
At 4 months of diet, the venous blood taken from the retro-orbital
plexus was collected on 0.138 M tri-sodium citrate 9/1 (vol/vol).
Within 10 min, the whole blood containing anticoagulant was
centrifuged at 1000g for 20 min and plasma (upper portion) was
immediately separated and frozen to −70 1C until examination.
Plasma concentrations of VEGF and SDF-1α were assayed by ELISA
(R&D Systems, Minneapolis, MN, U.S.A.). In brief, a monoclonal
antibody specific for VEGF and SDF-1α was pre-coated on to a
microplate. Standards and samples were pipetted into the wells,
and any VEGF and SDF-1α present was bound by the immobilized
antibody. After washing unbound substances, enzyme-linked poly-
clonal antibodies specific for VEGF and SDF-1α were added to the
wells. Following a wash to remove any unbound antibody–enzyme
reagent, a substrate solution was added to the wells and the colour
developed in proportion to the amount of VEGF and SDF-1α bound in
the initial step. Colour development was then stopped, and the
intensity of the colour was measured using a microplate reader set at
A450 nm.
2.8. Identification of proteins exposed on the surface of MPs and EPCs
MPs (1
104/ml) and EPCs (1
104/ml), from control group and
HH and HHI hamsters were sorted by flow cytometry in 1.5 ml
PBS and were examined again by flow cytometry in order to
identify and quantify the membrane receptors TF (Tissue factor),
P-Selectin, E-Selectin, PSGL-1 (P-selectin glycoprotein ligand-1),
Rantes (Chemokine (C–C motif) ligand 5, also CCL5) on the surface
of MPs and β2-Integrins (CD18/CD11) and α4β1-integrin on the
surface on EPCs.
Plasma MPs and EPCs were separated according to the method
reported by Georgescu et al. (2009, 2012).
In addition, dead cells were identified by staining with propi-
dium iodide (4 μg/ml) and controls experiments were made using
Dual Colour Control Reagent IgG-FITC/IgG1-PE. To take TF on MPs
29
as an example, the percentage for dead cells was  0.53 70.09%
and for isotype control  1.197 0.25%. Anyway, the results corre-
sponding to the rest of receptors on MPs and EPCs were
reproducible.
For flow cytometry technique, sorting speed was around
10,000–12,000 MPs, or EPCs per second. The experiments were
conducted using the Flow cytometer MoFlo (Dako, USA) equipped
with high-speed cell sorter.
2.9. Western-blotting analysis
The mesenteric arteries isolated from all groups of hamsters were
collected in PBS and minced into very small pieces after removing
connective tissue. The pieces were kept on ice and homogenized in
lysis buffer containing: 10 mM Trisma-base, 5 mM EDTA, 10% Triton
X-100, 25 μM PMSF, 1 μM benzamidine. The membrane proteins were
pelleted by centrifugation for 15 min at 9600g. The protein concen-
tration was assessed by the Bradford's method, and proteins (50 μg/
lane) were separated by SDS-PAGE. High range molecular weight
markers (6.5–205 kDa) were loaded into one lane as a size standard.
Gels were subsequently transferred to a nitrocellulose membrane. The
membranes were washed in 0.05% Tween 20 in PBS, and blocked in
5% BSA in PBS for 2 h. Afterwards, the membranes were incubated
overnight at 4 1C with the specific first antibodies. Next, membranes
were washed and incubated for 1 h with goat anti-rabbit IgG coupled
with peroxidase (HRP). The bound antibodies were detected in the
presence of ECL (Enhanced Chemiluminescent Substrate for detection
of HRP). The intensity of bands of proteins on blots was evaluated
using TL100 1D computer program from Nonlinear Dynamics (New-
castle upon Tyne, UK). The first antibodies were chosen for the
studied molecules: SDF-1α, VEGF, VEGFR1, VEGFR2, CXCR4, Tie2
(Angiopoietin-1), PIGF (Placental growth factor), COX-2 (Cyclooxygen-
ase-2), PGI2 synthase (Prostacyclin synthase) and β-actin.
2.10. Reagents
The standard chemicals, reagents and the specific antibodies were
purchased from Sigma Chemical Co. (St. Louis, MO, USA), Santa Cruz
Biotechonolgy (USA), and from Abcam (http://www.abcam.com,
USA). All others reagents used were of analytical grade.
2.11. Data analysis
NIS-Elements BR software was used to quantify the MP and EPC
infiltration in the vascular wall. The tension developed by arteries
was expressed as active wall tension (mN mm−1 arterial length)
and the relaxation was pondered as % of noradrenaline induced
contraction. Half-maximal concentrations (EC50) were calculated
from the dose–response curves. All values were expressed as
mean 7S.E.M. One-Way Analysis of Variance (abbreviated one-
way ANOVA) was used to compare means of two or more samples
between two different experimental groups (HH versus C, HHI
versus C or HH versus HHI). The means were considered signifi-
cantly different when P o0.05.
3. Results
3.1. The analysis of plasmatic parameters; the efficacy of
irbesartan treatment
The levels of standard parameters for atherosclerotic disease were
presented in our first papers on this model (Alexandru et al., 2011;
Georgescu et al., 2012). It was found that, the glucose concentrations
were not significantly different in the three investigated groups,
whereas the final weight, cholesterol and triglyceride concentrations,
30 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35
systolic and diastolic blood pressure and heart rate were significantly
increased for HH group compared to C (Po0.05). Also, the irbesartan
administration significantly reduced the levels of serum total choles-
terol, triglyceride, systolic and diastolic blood pressure and heart rate
which were augmented in HH model (Po0.05). Even though the
values for cholesterol and triglyceride concentrations were signifi-
cantly lower in the irbesartan treated animals compared to the HH
group, these were still several folds higher than C group ( 3 fold for
cholesterol and  2 fold for triglyceride) (Georgescu et al., 2012).
Fig. 2 shows the representative recordings of arterial blood
pressure and heart rate for the three experimental groups. The
values in mmHg for systolic blood pressure were 87.285 71.625
for C group, 120.89 70.63 for HH and 87.57 71.94 for HHI, while
for the diastolic blood pressure were 59.0773.39 for C group,
100.08 74.47 for HH and 72.66 73.84 for HHI. For the heart rate,
the values in bmp were 35971 for C group, 480.33714.83 for HH
and 353.57 716.76 for HHI.
3.2. MP and EPC infiltration in arterial vascular wall; the role of
irbesartan administration
The quantitative analysis of immunohistochemistry color
images developed significant changes in vascular wall of HH ham-
sters. The arterial lesions (showed in our previous paper
Georgescu et al., 2012) containing numerous MPs (labeled with
Annexin V-FITC) in HH group and EPCs (labeled with CD 34-PE) in
HHI group were identified (Fig. 3A and B). On the contrary, arterial
wall of HHI and C animals exhibited little or no MPs, while EPC
expression was reduced for HH group (Fig. 3A and B). However, the
analysis of all these changes revealed that irbesartan treatment
had a significant positive impact on EPC mobilization in vascular
wall. To quantify changes in stained areas after antibody binding,
the thoracic aorta and resistance artery sections from 6 hamsters
in each experimental group were analyzed with NIS-Elements
BR software in independently experiments. The above described
Fig. 2. The representative recordings of arterial blood pressure and heart rate in experimental groups: C, HH, HHI.
immunofluorescence images correlate very well with MP levels
(Annexin V-FITC positive) in plasma collected from C, HH, HHI
groups. MP quantification by flow cytometry analysis showed that
MP formation in HH group was higher then in C and HHI groups
(Table 1). Of interest, the level of circulating MPs did not remain
higher after irbesartan administration.
3.3. The endothelial function of arterial vascular wall; the beneficial
effect of irbesartan
Fig. 4 shows the effect of acetylcholine (10−8–10−4 M), an
endothelium-dependent vasodilator, on arterial rings explanted
from C, HH and HHI experimental groups. Acetylcholine relaxed
the arterial wall in a concentration-dependent manner, and by
using wire myograph technique the isometric force measurements
revealed significant decreases in the relaxation responses at HH
hamsters compared to C. The maximum amplitude of vasodilation
induced by acetylcholine was 16.73 74.57% of noradrenaline
induced contraction for HH arteries, while for C group was
64.19 75.23% (P o0.05, n ¼30) (Fig. 4).
Since AT1 receptor overexpression may play a decisive role in
the reduction of vasodilator response recorded at HH arteries, we
examined the effects of irbesartan treatment, an AT1 receptor
blocker, on the presence of endothelial dysfunction in HH group
(n ¼30). The developed force by arterial wall of HH hamsters
treated with irbesartan (HHI group) was increased compared to
that developed by arteries in HH group, untreated with irbesartan.
In this case, the maximum relaxation of HHI arteries was
57.86 7 4.91% of noradrenaline induced contraction, a value sig-
nificantly increased compared to that from HH arteries by
16.73 74.57% (Po 0.05, n ¼30) (Fig. 4), but very closed to that
from C arteries. This demonstrates that irbesartan ameliorates
the vascular dysfunction in term of endothelium-dependent
relaxation.
 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35 31

Fig. 3. Fluorescence microscopy of MPs (a) and EPC (b) in the vascular wall of thoracic aorta (A) and mesenteric resistance arteries (B) explanted from C, HH, HHI hamsters;
deparaffinized thin sections were incubated for 2 h with specific antibodies Annexin V-FITC (for MPs) and CD34-PE (for EPC). The results showed: (1) a lot of MPs (stained in
green) in HH group (area  8868.57 190.5 for aortic arch and  2524.57 65.5 for resistance arteries) and increased levels of EPCs (stained in red) in HHI group
(area  3112.75 775.25 for aortic arch and 2089.50 7 52.17 for resistance arteries); (2) very little MPs for HHI (area  1588.25 7 61.25 for aortic arch and 602.157 33.18 for
resistance arteries) and reduced levels of EPCs for HH group (326.8 719.2 for aortic arch and 271.3 7 15.22 for resistance arteries). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)

Table 1
The levels of circulating MPs in plasma from C, HH, HHI experimental groups by flow cytometry analysis.

MPs (Annexin V-FITC positive) C (n¼40) HH (n¼40) HH I (n¼ 40) P values (for HHI compared to HH)

MP/μl plasma 389.92 7 25.84 2967.257 7385.27 497.01 731.52 0.002

3.4. Measurement of plasma VEGF and SDF-1α; the effect

of irbesartan

Plasma levels of VEGF and SDF-1α were assayed by ELISA

method from venous blood collected from 30 hamsters in each
investigated experimental group. Table 2 shows significant eleva-
tions of VEGF levels (  2.33 fold) and SDF-1α (  2.72 fold) at HH
group compared to C (P o0.05). At HHI group, the irbesartan
administration for 4 month prevented the increases of these
parameters, keeping them close to values measured in plasma
collected from C group (Table 2).

3.5. Mechanistic insights in EPC homing to sites of

damaged endothelium

EPC recruitment and incorporation in the vascular wall requires
a coordinated sequence of multistep adhesive and signalling
events including adhesion and migration, chemoattraction and
finally the differentiation to endothelial cells. First of all, in the
three experimental groups we investigated the presence of β2-
Integrins (CD18/CD11) and α4β1-integrin on the surface of EPCs
(1
104/ml) positive for both hematopoietic stem cell markers,
such as CD34, and an endothelial marker protein as VEGFR2. Flow
cytometric analysis showed that the number of membrane recep-
tors CD18, CD11 and α4β1-integrin on the surface of EPCs was
significantly higher (  3 fold) in HH group than in C group
Fig. 4. The vasorelaxant function of C, HH and HHI resistance arteries to acetylcho-
line, an endothelium dependent vasodilator agent; the developed tension by
arteries was evaluated by isometric measurements.
(P o0.05) (Table 3). In addition, the irbesartan administration at
HHI group reduced the expression of these integrins until control
values (Po0.05) (Table 3).
Next, in an attempt to explore the mechanism involved in EPC
homing to arterial wall we determined by Western-blotting
technique the protein expression of pro-inflammatory endothelial
cytokines SDF-1α, VEGF, VEGFR1, VEGFR2, CXCR4, Tie2, PIGF in the
32 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35
Table 2
The quantification of plasmatic parameters: the vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 α (SDF-1α), for C, HH, HHI hamsters by ELISA
method.
Plasmatic parameters C (n¼ 40)
VEGF (pg/nl) 80.55 76.3
SDF-1α (ng/ml) 597.5 734.06
Table 3
The evaluation on membrane receptors β2, α4β1-integrin on the surface on EPCs as cells positive for CD34 and VEGFR2.
Integrins C (n ¼10)
% CD 18 25.617 2.11
% CD 11 22.45 7 1.98
% α4β1-integrin 18.727 1.79
vascular wall of mesenteric resistance arteries. Quantitative ana-
lysis indicated that the expressions of SDF-1α, VEGF, VEGFR1,
VEGFR2, CXCR4, Tie2, PIGF were significantly elevated in HH
arteries when compared with the control (Po0.05, n ¼8) (Fig. 5a
and b). The increased expressions of all these pro-inflammatory
endothelial cytokines were diminished after 4 month of irbesartan
administration in HHI group (Po 0.05) (Fig. 5a and b).
3.6. Mechanistic insights in MP adhering at vascular wall
The adhering capacity of MPs to arterial vascular wall was
explored by studying the pro-thrombotic activity of MPs as
microvesicles positive for Annexin V.
By flow cytometry analysis, we identified and quantified the
membrane receptors (TF, P-Selectin, E-Selectin, PSGL-1, Rantes) on
the surface of sorted MPs (1
104/ml) from HH group compared with
C and HHI groups (Table 3). In HH group, MPs were not only positive
for TF, P-Selectin, E-Selectin, PSGL-1, Rantes, but also a marked
increase ( 3 fold ) of their expression was observed when compared
with the control and HHI groups (Po0.05, n¼ 10) (Table 4).
As well, Western-blotting analysis showed that hypertension-
hyperlipidemia down-regulated the protein expression of COX-2
and PGI2 synthase molecules when compared with the control
and HHI groups. (P o0.05, n ¼8) (Fig. 6a and b). Notably, irbesartan
treatment increased the protein expression of COX-2, PGI2
synthase molecules that contribute to modulate the improvement
of arterial wall vasodilatation towards control values (P o0.05)
(n ¼8) (Fig. 6a and b).
4. Discussion
Atherosclerosis is now known to be a dynamic and progressive
process proceeding from the endothelial dysfunction and inflam-
mation of vascular wall. The evolution and prognosis of athero-
sclerosis, along with the efficacy of therapeutic modalities was
assessed by measuring the circulating levels of various biomarkers
expressed or released by the endothelium. Given their significant
biological contribution in different types of vascular pathologies,
EPCs and their biology have been under intense investigation for
the last decade (Caiado and Dias, 2012).
The present data demonstrate that treatment with AT1 receptor
blocker irbesartan in hypertensive–hypercholesterolemic hamster
model (HH) prevented atherosclerotic process that was associated
with increases in plasma MP levels, MP adhering at vascular wall and
decreases in EPC home in activated tissues in response to chemokines
HH (n¼ 40) HHI (n¼ 40) P values (for HHI compared to HH)
188.34 7 15.9 120.3 7 6.84 0.03
1628.75 7 345.73 893.75 7 127.98 0.004
HH (n¼10) HHI (n¼ 10) P values (for HHI compared to HH)
81.127 4.55 27.55 7 2.63 0.02
76.43 7 4.15 25.36 7 1.88 0.03
68.327 3.75 19.92 7 1.17 0.01
gradients that are formed in the tissue regions undergoing active
remodeling. The elevation of plasma/tissue VEGF, SDF-1α has been
noted in group with hypertension–hypercholesterolemia or athero-
sclerosis. In addition, MPs and VEGF/SDF-1α chemokines can been
regarded as a marker for the presence endothelial dysfunction
connected with the diminishment of endothelium-dependent vasodi-
lation. However, the role of irbesartan in relationship with EPC
mobilization, the improvement of endothelial function and the
mechanisms underlying these responses in atherosclerosis have not
been studied.
The major finding of this study is that in the experimental
hypertension associated with hypercholesterolemia, the treatment
with irbesartan significantly increased EPC homing and decreased
MP infiltration generating in this way the reestablishment of endothe-
lial function as a consequence of the increases of endothelium-
dependent relaxation to acetylcholine. However, to our knowledge,
the current study is the first one that analyzed the effect of irebesartan
on EPC/MP mobilization and homing to macro- and microvascular
wall in atherosclerosis. These beneficial effects of irbesartan are
strengthened by plasma levels of MPs and VEGF/SDF-1α, which were
significantly lower in HHI group when compared with HH hamsters.
Our results indicate that the elevation of plasmaVEGF/SDF-1α could be
associated with hypertension–hypercholesterolemia and the degree of
endothelial dysfunction. These data are in line with previous reports
indicating that plasma VEGF could be used as a marker of early
vascular damage induced by hypertension (Tsai et al., 2005). Also, the
elevation of VEGF was associated with atherosclerosis, but not diabetes
itself (Blann et al., 2002). All of the evidences, as well as our
observations, support the finding that elevation of VEGF levels is
associated with vascular atherosclerosis caused by various risk factors.
Regarding the reduction of MP levels after irbesartan treatment, this
could be caused by the specific effects of irbesartan. This explanation is
sustained by a paper which demonstrated that, in (ApoE)_/_ hyperlipi-
demic mice, Ang II infusion increases plasma endothelial microparticle
levels in a redox-sensitive, blood pressure—independent manner
(Burger et al., 2011). On the other hand, there are results which differ
from those of others implicating shear stress and blood pressure in MP
formation (Nomura et al., 2004; Labios et al., 2004). Moreover, our
results showed that, in hypertension associated hypercholesterolemia
the isolated MPs were characterized by an increased number of
specific membrane receptors TF, P-Selectin, E-Selectin, PSGL-1, Rantes,
while EPCs were depicted by a reduced number of β2-Integrins, α4β1-
integrin receptors, both of these being correlated with vascular
dysfunction of resistance arteries. After the irbesartan treatment for
4 month the number of specific receptors for MPs was reduced and for
EPCs was increased, contributing in this way to decreases in MP
 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35 33

Fig. 5. The exploration of mechanistic insights in EPC homing to sites of the damaged endothelium by studying the protein expression of pro-inflammatory endothelial
cytokines: SDF-1α, VEGF, VEGFR1, VEGFR2 CXCR4, Tie2, PIGF. (a) Representative Western blott of the mesenteric resistance arteries explanted from HH and HHI hamsters
compared to C hamsters; (b) The diagram of densitometric measurements: n¼ 8 individual experiments; protein expressions were considered as 100% for C group (nP o0.05
for HH versus C and HHI).

adhering at vascular wall and increases in EPC home at tissue lesion
followed by the improvement of vascular function. Furthermore, in an
attempt to explore the mechanistic insights in EPC homing to sites of
damaged endothelium we discovered the positive role of pro-
inflammatory endothelial cytokines VEGFR1, VEGFR2, CXCR4, Tie2,
PIGF, which were diminished after irbesartan administration. Also, our
data revealed the increase of protein expression of COX-2, PGI2
synthase molecules with role in the improvement of arterial wall
vasodilatation as a consequence of the treatment with irbesartan. In
addition, in accord with our results, it has been demonstrated that the
consequences of PGI2 suppression, elevation of blood pressure, accel-
eration of atherogenesis and modulation of the vascular response to
hemodynamic stress, may converge to alter vascular architecture and
elevate cardiovascular risk during extended dosing with selective
inhibitors COX-2 (Rudic et al., 2005). A possible explanation for our
experimental data could be that, in atherosclerosis MPs harboring
some functional proteins as TF, P-Selectin, E-Selectin, PSGL-1, Rantes
induce pro-inflammatory effects on endothelial cells and the vascular
wall and can stimulate the release of pro-inflammatory endothelial
cytokines, includingVEGFR1, VEGFR2, CXCR4, Tie2, PIGF and induce the
34 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35

reduction in the expression of COX-2, PGI2 synthase molecules. In
addition, our results revealed the possibility of regulating pro-
inflammatory properties of circulating MPs through irbesartan treat-
ment. However phenotypic and functional characterization of MPs
should be considered a key step in an attempt to understand their role
in endothelial and vascular homeostasis. In a previous study, it was
promoted the idea that platelet-derived microparticles (PMP) might
contribute to the homing of EPCs, to angiogenesis and vascular repair
(Morel et al., 2009). In our study we showed that circulating EPCs
provide protection against atherosclerosis by their innate ability to
regenerate damaged endothelium and contribute to the maintenance
and regeneration of plaque endothelium in the HH hamster by their
membrane receptors β2-Integrins, α4β1-integrin. Also, we give evi-
dences that irbesartan therapy may provide an important strategy to
enhance EPC number, mobilization, survival, engraftment, and func-
tion, thereby rendering these cells efficient therapeutic modalities for
cardiovascular diseases. Numerous studies concerning EPC biology and
function indicate that integrins represent a major molecular determi-
nant of EPC function, with different integrin subunits regulating
different steps of EPC biology (Caiado and Dias, 2012). Particularly,

Table 4
The estimation of pro-thrombotic activity of MPs (as microvesicles positive for Annexin V) by quantification of specific receptors (TF, P-Selectin, E-Selectin, PSGL-1, Rantes) on
their surface by flow cytometry analysis.

Receptors on MPs C (n¼10) HH (n¼10) HHI (n¼10) P values

(for HHI compared to HH)

% TF 28.217 2.15 73.79 7 4.02 30.117 2.56 0.01

% P-Selectin 33.867 2.75 68.597 3.45 37.137 2.82 0.02
% E-Selectin 32.52 7 2.15 65.917 3.82 35.227 2.36 0.03
% PSGL-1 26.117 2.17 52.23 7 2.98 29.147 2.77 0.01
% Rantes 22.52 7 2.44 45.137 2.76 25.92 7 2.52 0.02

Fig. 6. The quantification of COX-2, PGI2 synthase molecule expression with role in the improvement of arterial wall vasodilatation after EPC attachment. (a) Representative
Western Blott of the mesenteric resistance arteries explanted from HH and HHI hamsters compared to C hamsters; (b) The diagram of densitometric measurements:
n¼ 8 individual experiments; protein expressions were considered as 100% for C group (nP o0.05 for HH versus C and HHI).
 A. Georgescu et al. / European Journal of Pharmacology 711 (2013) 27–35
integrin α4β1 could be a key regulator of EPC retention and/or
mobilization from the bone marrow, while integrins α5β1, α6β1 major
determinants of EPC homing, invasion, differentiation and paracrine
factor production. Also, β2 integrins are the major regulators of EPC
transendothelial migration. The relevance of integrins in EPC biology is
also demonstrated by any studies that use extracellular matrix-based
scaffolds as a clinical tool to improve the vasculogenic functions of
EPCs (Critser et al., 2010; Hanjaya-Putra et al., 2010). Thus, EPC by
their integrin profile can adapt to different conditions and can
influence the function of other cells with important role in the
vascular atherosclerosis.
5. Conclusions
Taken together, our results suggest that: (1) increased levels of
circulating MPs and reduced levels of circulating EPCs dependently
predict atherosclerotic disease progression; (2) MPs within the arterial
wall may be associated with lesion progression to atherosclerotic
plaque formation by the presence on their plasma membrane of TF,
P-Selectin, E-Selectin, PSGL-1, Rantes proteins; (3) EPCs localized in
proximity to arterial vascular injury may contribute to on-going
process of endogenous endothelial repair by the participation of β2-
Integrins, α4β1-integrin proteins; (4) irbesartan administration
enhances EPC homing and decreases MP infiltration to sites of
damaged endothelium contributing to improvement of endothelial
function as a consequence of the increases of endothelium-dependent
relaxation to acetylcholine; irbesartan effects are strengthened by
plasma levels of VEGF/ SDF-1α; (5) irbesartan administration improves
EPC/MP number and functionality by various mechanisms generating
the modulation of atherosclerosis progression, the rebalancing
between endothelial injury and repair; pro-inflammatory endothelial
cytokines VEGFR1, VEGFR2, CXCR4, Tie2, PIGF, and COX-2, PGI2
synthase molecules have the positive role in this process.
In conclusion, our data provide evidences for the hypothesis that
irbesartan increases the mobilization of EPCs in atherosclerosis by an
inhibitor effect on circulating MPs and contributes to ongoing vascular
repair. Thus, the regeneration of dysfunctional vascular wall which
represents the common trunk for all cardiovascular diseases may be
important for choosing therapy strategies with a maximized benefit
for the patient and irbesartan treatment appears as an appealing
challenge in the treatment of complex vascular atherosclerosis.
Moreover, we think that the manipulation of MP and EPC
behavior of their specific interactions with vascular wall layers
and plaque components may be crucial to further improve the
usage of these cells as relevant clinical agents.
Acknowledgements
The authors appreciate dedicated work of Marilena Isachi,
Marcela Toader (biochemistry), Sanda Nae (experimental model)
and of Elena Florea, Ana Manole, Marilena Misici (microscopy).
This work was supported bby grants of the Romanian National
Authority for Scientific Research, CNCS – UEFISCDI, project ID PNII-CT-
ERC-2012–1 (6ERC- like/July 18, 2012), project ID PN-II-ID-PCE-2012-
4-0124, project PN-II-ID-PCCE No.248/2010, by European Social
Fund – ‘Cristofor I. Simionescu’ Postdoctoral Fellowship Programme
(ID POSDRU/89/1.5/S/55216), Sectoral Operational Programme Human
Resources Development 2007–2013, and by Romanian Academy.
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