Atherosclerosis 242 (2015) 167e173

Contents lists available at ScienceDirect

Atherosclerosis
journal homepage: www.elsevier.com/locate/atherosclerosis

Metformin modulates human leukocyte/endothelial cell interactions

and proinammatory cytokines in polycystic ovary syndrome patients
~ uls a, b, 1,
Victor M. Victor a, b, c, d, *, 1, Susana Rovira-Llopis a, b, 1, Celia Ban
 pez a,
Noelia Diaz-Morales a, Sandra Lopez-Domenech a, Irene Escribano-Lo
a, d
d, e
a, b
, Angeles Alvarez
, Marcelino Gomez
, Milagros Rocha a, b, d, ***,
Cesar Rios-Navarro
a, b, f, **
Antonio Hernandez-Mijares
a

Service of Endocrinology, University Hospital Dr. Peset, Foundation for the Promotion of Health and Biomedical Research in the Valencian Region
(FISABIO), Valencia, Spain
b
Institute of Health Research INCLIVA, University of Valencia, Valencia, Spain
c
Department of Physiology, University of Valencia, Valencia, Spain
d
Department of Pharmacology and CIBERehd, Faculty of Medicine, University of Valencia, Spain
e
n General de Universidad de Valencia, Valencia, Spain
Fundacio
f
Department of Medicine, University of Valencia, Valencia, Spain

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 7 April 2015
Received in revised form
17 June 2015
Accepted 7 July 2015
Available online 10 July 2015

Objective: We aim to assess the effect of metformin treatment on metabolic parameters, endothelial
function and inammatory markers in polycystic ovary syndrome (PCOS) subjects.
Methods: The study population consisted of 40 reproductive-age women with PCOS, who underwent
treatment with metformin during a 12-week period, and their corresponding matched controls (n 44).
We evaluated endocrinological parameters, adhesion molecules (vascular cell adhesion molecule 1
(VCAM-1), intercellular cell adhesion molecule 1 (ICAM-1) and E-selectin) and proinammatory cytokines (interleukin 6 (IL-6) and tumor necrosis factor alpha (TNFa)) in serum. In addition, interactions
between human umbilical vein endothelial cells and polymorphonuclear (PMN) cells were assessed by
ow chamber microscopy. In addition, a group of type 2 diabetes patients who underwent treatment
with metformin during a 12-week period was incorporated into the study.
Results: Metformin produced benecial effects on PCOS patients by decreasing polymorphonuclear
(PMN) rolling ux and adhesion. It also decreased levels of ICAM-1, E-selectin, IL-6 and NFa. In addition,
metformin induced an improvement of endocrine and anthropometric parameters in PCOS subjects by
reducing glucose, follicle-stimulating hormone (FSH) and androstendione, and by increasing
dehydroepiandrosterone-sulfate (DHEA-S). Metformin also had benecial effects in type 2 diabetic
subjects by reducing body weight, waist circumference and PMN adhesion, and by increasing PMN
rolling velocity.
Conclusion: Our results highlight the modulating effect of metformin on leukocyte/endothelium interactions. These ndings may explain the potential benecial effect of metformin in reducing the risk of
vascular events in PCOS patients and in insulin resistance conditions.
2015 Elsevier Ireland Ltd. All rights reserved.

Keywords:
Metformin
Mitochondria
Leukocyte
Endothelium
PCOS
Type 2 diabetes

1. Introduction
* Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
** Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
*** Corresponding author. University Hospital Doctor Peset, Avda Gaspar Aguilar
90, 46017, Valencia, Spain.
E-mail addresses: Victor.Victor@uv.es (V.M. Victor), Milagros.Rocha@uv.es
(M. Rocha), hernandez_antmij@gva.es (A. Hernandez-Mijares).
1
These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.atherosclerosis.2015.07.017
0021-9150/ 2015 Elsevier Ireland Ltd. All rights reserved.

Polycystic ovary syndrome (PCOS) is a metabolic disorder

characterized by the presence of hyperandrogenism, oligoovulation or anovulation and polycystic ovaries [1]. A high incidence of insulin resistance (IR) and cardiovascular risk factors such
as hypertension, obesity, dyslipidemia and type 2 diabetes are reported in PCOS subjects [2,3], all of which increases the probability

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V.M. Victor et al. / Atherosclerosis 242 (2015) 167e173

of future cardiovascular disease. In addition, several studies have

indicated that PCOS subjects can develop subclinical atherosclerosis at a young age [4e6], while others have reported increased
cardiovascular morbidity in these patients, though the available
data are inconclusive and scarce [7,8].
Endothelial dysfunction occurs at the beginning of the atherosclerotic process [9] and is related to cardiovascular events [10]. In
this sense, different studies have demonstrated that PCOS subjects
can develop endothelial dysfunction [4,5], and this dysfunction has
been shown to be related to IR and/or hyperandrogenism [4,11].
Accumulation of leukocytes in the vessel wall is a hallmark of
the early stages of atherosclerosis and vascular disease, and is
mediated by interaction between the adhesion molecules
expressed on white blood and/or endothelial cells. Different cellular
adhesion molecules have been implicated in the atherogenic process, including vascular cell adhesion molecule-1 (VCAM-1),
intercellular adhesion molecule-1 (ICAM-1) and selectins [12].
During this process, leukocytes roll along the wall of inamed
vessels before coming to a halt, after which they adhere and
transmigrate [13].
Several studies in PCOS patients have demonstrated that acute
hyperglycemia and IR induce an increase in reactive oxygen species
(ROS) production by peripheral blood leukocytes [14,15], activation
of leukocyteeendothelium interactions [16] and the proinammatory transcription factor nuclear kB (NF-kB) [17], and an
increase of proinammatory cytokines [18]. In this way, oxidative
stress and activation of leukocyte/endothelium interactions and
inammation have been implicated in the etiology of IR in the
leukocytes of PCOS patients.
Insulin sensitizers such as metformin are widely used in the
treatment of PCOS because of their capacity to improve IR [19],
ovulation [20] and hyperandrogenism [21]. In addition, it has been
speculated that metformin improves endothelial function in PCOS
subjects [22], although conicting results have been published
regarding its effects on ow-mediated dilation in these patients
[21,23].
The present study set out to explore the effects of metformin
treatment on leukocyte/endothelium interactions, adhesion molecules and proinammatory cytokines in PCOS patients by demonstrating their benecial effects on rolling ux, PMN adhesion,
ICAM-1, E-selectin, IL-6 and TNFa levels. Moreover, as expected,
metformin induced an improvement of endocrine and anthropometric parameters in PCOS subjects. Metformin also had benecial
effects in type 2 diabetic subjects by reducing body weight, waist
circumference, PMN adhesion, and by increasing PMN rolling
velocity.
2. Materials and methods
2.1. Subjects
Forty women of fertile age (18e40 years old) diagnosed with
PCOS and forty-four matched controls were enrolled in the study
(Table 1). The controls were volunteers recruited from the University Hospital Dr. Peset and the Faculty of Medicine (University of
Valencia), and were selected according to age and body mass index
(BMI). Control subjects had regular menses, levels of testosterone
lower than 0.9 ng/ml, and no family history of PCOS, diabetes or
familial combined hyperlipidemia.
PCOS subjects were diagnosed according to the Rotterdam
criteria [1] and were drawn from a larger patient cohort that
participated in another study conducted by the Department of
Endocrinology, University Hospital Doctor Peset, Valencia, Spain.
The aforementioned criteria for the diagnosis of PCOS included
elevated free testosterone levels (>0.5 ng/dl; the cut-off level for

free testosterone was the mean 2 SD according to normal levels in

controls), hirsutism (total Ferriman-Gallwey score>7), oligoovulation (cycles longer than 35 days or shorter than 26 days) [24] and
polycystic ovaries (identied by transvaginal ultrasonography; the
presence of 12 or more small (2e9 mm) follicles in each ovary).
Ultrasound scans were performed and scored independently by
one of two experienced and blinded reviewers. None of the subjects
had galactorrhea or any endocrine or systemic disease that could
have affected her reproductive physiology. None of the women
reported using any medication that could have interfered with the
normal function of the hypothalamic-pituitary-gonadal axis during
the previous semester. Exclusion criteria were haematological, infectious, organic, malignant, or inammatory disease, or a history
of stroke, ischemic heart disease, or thromboembolism and diabetes mellitus.
PCOS subjects and controls followed a normocaloric diet. In the
PCOS group, treatment with metformin was initiated at 500 mg per
day and increased to 1000 mg/d after 2 weeks, and then to
1500 mg/d after a further 2 weeks, and was maintained at this dose
for a total of 12 weeks. No intolerance to the metformin treatment
was detected. Subjects were seen every two weeks for endocrinological and anthropomorphic measurements and blood sampling.
Compliance was determined by questioning subjects during their
fortnightly visits. Informed written consent was obtained from all
subjects prior to participation.
In addition to the abovementioned groups, 38 patients with
type 2 diabetes were recruited and received metformin treatment
over a total period of 12 weeks. Type 2 diabetes was diagnosed in
these subjects according to the criteria established by the American
Diabetes Association (ADA) [25].
The study was approved by the ethics committee of the Hospital
Dr. Peset and was performed in accordance with the Helsinki
declaration. This trial was registered on clinicaltrials.gov under
study number NCT02302326.
An anthropometric and analytical evaluation was performed,
and height (m), weight (kg) and waist (cm) measured in all subjects. Body mass index (BMI weight (kg)/height (m)2) was then
calculated. Blood was collected from the antecubital vein at 8e10
a.m, after 12 h of fasting, during the follicular phase, on the second/
third day of the menstrual cycle. Insulin was measured by an
enzymatic luminescence technique. Glucose levels were measured
using enzymatic techniques and a Dax-72 autoanalyzer (Bayer
Diagnostic, Tarrytown, New York, USA). IR was calculated by homeostasis model assessment (HOMA) using baseline glucose and
insulin: HOMA (fasting insulin (mU/ml)  fasting glucose (mmol/
L)/22.5. LH, sex hormone binding globulin (SHBG), androstendione
and testosterone were measured by specic RIAs. Dehydro
epiandrosterone-sulfate (DHEAS) was measured by means of a
specic chemiluminescence technique. High sensitive C-reactive
protein (hsCRP) was quantied by a latex-enhanced immunonephelometric assay (Behring Nephelometer II, Dade Behring, Inc.,
Newark, DE, USA) with an intra-assay coefcient of variation of 8.7%
and sensitivity of 0.01 mg/L. Total cholesterol and triglycerides
were measured by employing enzymatic assays, and HDLc concentrations were recorded with a Beckman LX-20 autoanalyser
(Beckman Coulter, La Brea, CA, USA) using a direct method. The
intraserial variation coefcient was <3.5% for all determinations.
LDLc concentration was calculated using the Friedewald method.
Apolipoprotein (Apo) AI and B was determined by immunonephelometry (Dade Behring BNII, Marburg, Germany) with an intraassay variation coefcient of <5.5%.
2.2. Cells
Human polymorphonuclear leukocytes (PMNs) were obtained

V.M. Victor et al. / Atherosclerosis 242 (2015) 167e173

169

Table 1
Anthropometric parameters, lipoprotein prole, hydrocarbonated metabolism parameters and circulating androgens of control and PCOS women treated with metformin
during a 12-week period.
Controls (n 44)
Age (years)
Body weight (kg)
BMI (Kg/m2)
Waist circumference (cm)
Systolic BP (mmHg)
Diastolic BP (mmHg)
Total cholesterol (mg/dl)
LDLc (mg/dl)
HDLc (mg/dl)
Triglycerides (mg/dl)
Apolipoprotein A (mg/dl)
Apolipoprotein B (mg/dl)
hsCRP (mg/l)
Glucose (mg/dl)
Insulin (mIU/ml)
HOMA-IR
FSH (mIU/ml)
LH (mIU/ml)
Testosterone (ng/ml)
DHEA-S (mg/dl)
Androstendione (ng/ml)
SHBG (nmol/l)

26.6
77.6
29.3
86.9
109
70
174.2
106.1
51.6
83.7
142.6
80.6
2.87
86.1
9.7
2.14
4.34
5.51
0.43
231.1
2.49
103.8

4.5
17.5
6.6
16.9#
11#
10
31.6
26.8
13.3#
47.2
30.2
21.3
2.49#
10.2
5.3#
1.29#
2.40
5.11
0.20#
108.4#
0.96#
69.4#

PCOS baseline (n 40)

25.0
83.1
30.8
94.3
119
74
176.1
112.4
45.7
94.8
132.9
85.2
4.21
85.7
14.2
3.07
4.87
6.30
0.74
342.1
3.96
52.4

7.2
23.1
8.6
17.0
16
12
32.6
27.0
10.0
55.0
18.8
23.3
3.11
10.1
8.8
2.06
1.50
4.88
0.39
170.2
1.68
36.6

PCOS 12 weeks (n 40)

e
82.8
30.6
94.4
117
73
172.5
108.1
45.2
101.1
132.4
84.2
3.93
81.7
13.2
2.76
4.23
5.94
0.68
386.2
3.44
42.9

23.1
8.6
15.9
16
10
32.1
26.9
7.7
61.4
15.1
26.0
3.52
10.1*
8.5
2.16
1.67*
4.17
0.36
200.2*
1.60*
21.9

Data are expressed as mean SD. # Statistical signicance (p < 0.05) was determined by an unpaired Student's t-test (control vs PCOS).* Statistical signicance (p < 0.05) was
determined by a paired Student's t-test (baseline vs 12 weeks).

from citrated blood samples and were incubated for 45 min with
dextran (3%). The supernatant was centrifuged at 250 g for 25 min
over Fycoll-Hypaque. Lysis buffer was added to the pellet, which
was centrifuged and washed twice at room temperature (100 g,
5 min). PMNs were evaluated in a Scepter device (Millipore, MA,
USA), washed in HBSS medium and stored in complete RPMI media
at 37  C.
2.3. Adhesion assay. Levels of cytokines and adhesion molecules.
Nitric oxide measurements
The parallel plate ow chamber in-vitro model has been
described in detail elsewhere [26,27]. A Luminex 200 ow analyzer
system was used to evaluate ICAM-1, VCAM-1, E-selectin, IL-6 and
TNF-alpha in serum from PCOS subjects (Millipore, Austin, TX,
USA).
Total nitric oxide (NO) in the serum samples was measured with
the total NO/Nitrite/Nitrate Parameter Assay Kit (R & D Systems,
Minneapolis, Minnesota, USA). Concentrations of NO were determined by Griess reaction, in which acidied NO2 produces a
nitrosating agent, which reacts with sulfanilic acid to form the
diazonium ion. This ion is then coupled to N-(1-naphthyl)ethylenediamine to produce the chromophoric molecule that absorbs
light at 540e570 nm.
2.4. Drugs and solutions
Glucose, trypan blue, arginine, TNFa, albumin and bronectin
were obtained from SigmaeAldrich. Dextran was acquired from
Fluka (St. Louis, MO). RPMI 1640 supplemented with 20 mm HEPES,
endothelial cell growth medium culture media, HBSS, DPBS with
(DPBS) or without (DPBS) Ca2 and Mg2, and fetal bovine serum
were obtained from Lonza (Verviers, Belgium). FicollePaque Plus
was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). Plastic coverslips with a diameter of 25 mm were
obtained from Nunc (supplied by Thermo Fisher Scientic), sodium
pyruvate and trypsin-EDTA were obtained from Invitrogen (Eugene,
OR). PBS and collagenase, were obtained from Gibco (Life Technologies, Eugene, Oregon).

2.5. Data analysis

Statistical analysis was performed with SPSS 17.0 software (SPSS
Statistics Inc., Chicago, IL, USA). Continuous variables were
expressed as mean and standard deviation (SD) or as median and
25th and 75th percentiles for parametric and non-parametric data,
respectively. The 12-week intervention period following completion of the metformin regimen in PCOS and type 2 diabetes was
evaluated using a paired Student's t-test and control vs PCOS and
control vs type 2 diabetes data were evaluated using an unpaired
Student's t-test. To minimize the potential inuence of BMI, analysis of covariance was performed. All the tests used a condence
interval of 95% and differences were considered signicant when
p < 0.05.
3. Results
3.1. Clinical and metabolic characteristics
The clinical and metabolic characteristics of PCOS subjects are
presented in Table 1. No differences were obtained between values
among controls at baseline and after 3 months (data not shown).
Metformin treatment decreased glucose (P < 0.05), FSH (P < 0.05)
and androstendione (P < 0.05) and increased DHEA-S levels
(P < 0.05). Supplementary Table 1 shows the clinical and metabolic
characteristics of type 2 diabetic patients in basal conditions and
after 3 months of metformin treatment. Metformin treatment
decreased body weight (P < 0.01) and waist circumference
(P < 0.01).
3.2. Adhesion assay under ow conditions
We observed a decrease in the rolling velocity of PMN (P < 0.01)
and an increase in rolling ux (P < 0.001) and adhesion (P < 0.01) in
PCOS patients, as we have reported previously [16]. In the present
study, we observed that metformin treatment did not affect PMN
rolling velocity (micrometers per second) (Fig. 1A), but that there
was a decrease in PMN rolling ux (cells per minute, P < 0.05)
(Fig. 1B) and adhesion (cells per square millimeter, P < 0.05) after 3

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V.M. Victor et al. / Atherosclerosis 242 (2015) 167e173

months of metformin treatment (Fig. 1C). These results show that

metformin decreases leukocyteeendothelium interactions in PCOS
patients. In addition, we observed a signicant decrease in PMN
rolling velocity (417.7 89.51 mm/s, P < 0.01) in diabetic patients
with respect to controls, a signicant increase in PMN rolling ux
(201.1 80.89 cells/min, P < 0.01) with respect to controls, and an
increase in PMN adhesion (12.87 6.79 cells/mm2, P < 0.05) with
respect to the corresponding control group. Treatment with metformin for 3 months signicantly increased PMN rolling velocity
(552.8 143.3 mm/s, P < 0.05) and decreased adhesion (10.65 5.14
cells/mm2, P < 0.05) with respect to basal values in the diabetic
group, while no statistical differences were detected in the case of
rolling ux (234.4 110.7 cells/min).
3.3. Levels of adhesion molecules, cytokines and NO

Regarding to the levels of cytokines, PCOS induced an increase of IL6 (Fig. 3A, P < 0.05) and TNFa (Fig. 3B, P < 0.05), while metformin
decreased IL-6 (Fig. 3A, P < 0.05) and TNFa (Fig. 3B, P < 0.05) thus
suggesting an anti-inammatory effect. Regarding levels of NO, no
statistical differences were detected in any of the study groups
(Fig. 4).

4. Discussion
The present study was designed to evaluate the effects of metformin treatment on leukocyte/endothelium interactions, adhesion
molecules and proinammatory cytokines in PCOS patients. We
have observed that metformin can exert benecial effects by
decreasing PMN rolling ux and adhesion, and ICAM-1, E-selectin,
IL-6 and TNFa levels. As expected, we have also seen that it induces

There was an increase in VCAM-1 (Fig. 2A, P < 0.05), ICAM-1

(Fig. 2A, P < 0.05) and E-Selectin (Fig. 2C, P < 0.01). Metfomin did
not modify levels of VCAM-1 (Fig. 2A), but did decrease levels of
ICAM-1 (Fig. 2B, P < 0.05) and E-selectin (Fig. 2C, P < 0.05).

Fig. 1. Effect of metformin treatment on PMN rolling velocity (mm s1) (A), rolling ux
(PMN per minute) (B), and PMN adhesion (PMN per square millimeter) (C). *P < 0.05;
**P < 0.01 and ***P < 0.001 vs. control group. aP < 0.05 vs. before treatment.

Fig. 2. Effect of metformin treatment on VCAM-1 (pg/mL) (A), ICAM-1 (pg/mL) (B), and
E selectin (pg/mL) (C). *P < 0.05 and **P < 0.01 vs. control group. aP < 0.05 vs. before
treatment.

V.M. Victor et al. / Atherosclerosis 242 (2015) 167e173

Fig. 3. Effect of metformin treatment on IL-6 levels (A) and TNFa levels (B); both were
evaluated by Luminex 200 ow analyzer system. *P < 0.05 vs. control group. aP < 0.05
vs. before treatment.

Fig. 4. Effect of metformin treatment on NO levels. Total nitric oxide (NO) in the serum
samples was measured with the total NO/Nitrite/Nitrate Parameter Assay Kit (R & D
Systems, Minneapolis, Minnesota, USA).

a benecial effect on endocrine and anthropometric parameters by

decreasing glucose, FSH and androstendione, and by increasing
DHEA-S levels. In addition, metformin demonstrated a benecial
effect in type 2 diabetes subjects by reducing body weight, waist
circumference, PMN adhesion, and by increasing PMN rolling
velocity.
PCOS is characterized by different cardiovascular risk factors,
such as hypertension, obesity, impaired glucose tolerance, and lipid
abnormalities [28]. In fact, several studies have shown an increased
prevalence of cardiovascular diseases and morbidity in PCOS

171

subjects [29]. In this sense, metformin has been shown to slow

down signicantly the progression of type 2 diabetes in patients
with impaired glucose tolerance (IGT) at baseline [30]. Metformin
has also been reported to decrease LDL and systolic blood pressure,
thus reducing the risk of atherogenesis and ameliorating metabolic
syndrome in PCOS subjects [30]. This previous evidence is in line
with our results demonstrating that some of the benecial effects
of metformin are achieved through a decrease in body weight, BMI,
LDL, glucose and HOMA-IR.
Leukocyte recruitment to the arterial wall is related to atherosclerosis and hypertension. In the present study, we have evaluated
this function by using a model which mimics the ow of leukocytes
over the endothelium [16,26]. This reproduces the processes that
precede the formation of an inammatory focus in vivo (rolling and
adhesion) and which are critical to haemostasis and vascular cell
integrity. However, an exacerbation of these interactions contributes to the vascular impairment related with vascular diseases (e.g.
atherosclerosis, hypertension) [31]. Our system has been widely
used to analyze the different steps of leukocyte/endothelium interactions, and allows the mechanisms of action implicated to be
identied [32]. In the present study, we have observed that metformin treatment has benecial effects by decreasing rolling ux
and PMN adhesion in PCOS subjects, which, in turn, may impede
the development of the atherogenic process. In addition, we have
demonstrated that metformin increases rolling velocity and decreases PMN adhesion in type 2 diabetic subjects. In relation with
this, metformin has been shown to improve biochemical and
functional markers of endothelial reactivity and to reduce brinolysis [33]. Metformin improves vascular function via attenuation of
IR/hyperinsulinemia [19], and can also decrease serum levels of
asymmetric dimethylarginine (ADMA), an endogenous inhibitor of
NO synthase, by modulating androgen levels and insulin action
[34]. In addition, metformin stimulates AMPK and phosphorylates
endothelial nitric oxide synthase (eNOS) [19,35], while it enhances
NO levels and diminishes Rho kinase in animal models of hyperlipidemia [36]. However, in the present study we have no detected
differences in NO levels between control and PCOS subjects either
in basal conditions or after metformin treatment. In this sense,
Kandarakis et al. (2007) demonstrated that metformin reduced
serum advanced glycation end products (AGEs), which are oxidative mediators [37]. Metformin has also been shown to have
benecial effects by improving arterial stiffness and endothelial
function in young women with PCOS [38]. On the whole, these data
support the idea that metformin can exert benecial effects on
endothelial function and leukocyte/endothelium interactions.
Endothelial activation can be assessed by measuring the soluble
adhesion molecules E-selectin, VCAM-1 and ICAM-1. These substances are up-regulated during the innate immune response and
participate in the recruitment of leukocytes to the site of infection.
Dysregulation of endothelial activation may be associated with
increased morbidity and susceptibility to infection [39]. In general,
levels of adhesion molecules are elevated in patients with type 2
diabetes mellitus [40] and are up-regulated in healthy individuals
in response to systemic inammation. In the present work, we have
observed that the increased levels of ICAM-1 and E-selectin adhesion molecules characteristic of PCOS subjects are lower after
treatment with metformin, in accordance with a reduction in leukocyteeendothelium interactions. Available data regarding the effect of metformin on adhesion molecules are contradictory; for
example, Bilgir et al. showed that treatment with ethinylestradiol
(EE)/cyproterone acetate (CA) plus metformin decreases levels of
VCAM-1 and ICAM-1 in PCOS [41], while Kandarakis et al. reported
that metformin treatment decreases levels of VCAM-1 only. These
discrepancies may be due to variations in the dose and/or duration
of metformin treatment in question [42].

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V.M. Victor et al. / Atherosclerosis 242 (2015) 167e173

Leukocytes also release proinammatory cytokines such as

TNFa and IL-6, which can play a key role in the appearance of IR in
PCOS patients [15]. In the present study, we have demonstrated
that metformin decreases TNFa and IL-6 levels, which would suggest a benecial, anti-inammatory effect. In addition, it has been
reported that metformin inhibits the activation of NF-kB in HUVEC
exposed to inammatory cytokines, thus reducing the expression
of genes that encode adhesion and proinammatory molecules
[43].
The number of patients in the different study groups, and the
fact that evaluation of fasting glucose levels does not necessarily
rule out abnormal glucose tolerance in PCOS are two potential
limitations of this study. In addition, our results do not throw light
on the mechanism by which metformin modulates human leukocyte/endothelial cell interactions and proinammatory parameters
under insulin resistance conditions, which needs to be explored in
future research.
In conclusion, the present study demonstrates that metformin
has benecial effects on leukocyte/endothelium interactions by
decreasing rolling ux and PMN adhesion and levels of ICAM-1, Eselectin, IL-6 and TNFa. Future studies should evaluate whether
treatment with insulin sensitizers can improve cardiovascular
function in PCOS subjects in particular and in conditions of insulin
resistance in general.
Conict of interest
The authors have declared no conicts of interest.
Acknowledgments
We thank Isabel Soria for her work in the extraction of biological
samples (University Hospital Dr Peset), and B Normanly for his
editorial assistance (University of Valencia). This study was funded
by grants PI12/1984, PI13/1025, PI13/0073, UGP14-093 and UGP14095 from FISABIO, CIBERehd CB06/04/0071, PROMETEOII 2014/035,
SAF2010/16030 and by the European Regional Development Fund
(ERDF A way to build Europe). V.M.V. and M.R. are recipients of
contracts from the Ministry of Health of the Valencian Regional
Government and Carlos III Health Institute (CES10/030 and CP10/
0360, respectively). S.R.-LL., N.D.-M., and S.L.-D. are recipient of a
predoctoral fellowship from Carlos III Health Institute (FI11/00637,
FI14/00125 and FI14/00350). C.B. is recipient of a Sara Borrell contract from Carlos III Health Institute (CD14/00043). C.R.N. was
n, Cultura y Deporte (CPI-13supported by Conselleria de Educacio
n
194). A.A. was supported by Ministerio de Ciencia e Innovacio
n y Cajal program RYC2005-002295 and I3 program).
(Ramo

[6]

[7]

[8]

[9]
[10]

[11]
[12]

[13]
[14]

[15]

[16]

[17]

[18]

[19]
[20]

[21]

[22]

[23]

[24]
[25]
[26]

Appendix A. Supplementary data

[27]

Supplementary data related to this article can be found at http://

dx.doi.org/10.1016/j.atherosclerosis.2015.07.017.

[28]
[29]

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