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Role of endothelial cell membrane transport in red wine

polyphenols-induced coronary vasorelaxation:
Cite this: Food Funct., 2013, 4, 1452
involvement of bilitranslocase
Lovro Ziberna,ab Jong-Hun Kim,b Cyril Auger,b Sabina Passamonti*a
and Valérie Schini-Kerthb
Published on 31 July 2013. Downloaded on 07/10/2013 10:39:15.

Received 2nd May 2013
Accepted 30th July 2013
DOI: 10.1039/c3fo60160a
www.rsc.org/foodfunction
Red wine polyphenols (RWP) induce nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-
mediated coronary vasodilatation involving the redox-sensitive PI3-kinase/Akt-dependent pathway in the
endothelium. However, there is a gap of knowledge in explaining how bioactive polyphenols initialize
their signalling pathway in endothelial cells. Here, we investigated the hypothesis that flavonoids act
subsequently to their entry into the endothelium via the flavonoid membrane transporter bilitranslocase
(TC 2.A.65.1.1). Thus, vascular reactivity studies were performed using isolated porcine coronary artery
rings. We separately determined the NO- and EDH-mediated components of the relaxation in the
presence of specific inhibitors. In either case, bilitranslocase antibodies significantly reduced the relaxations
of coronary artery rings induced by RWP. Furthermore, bilitranslocase antibodies significantly reduced
RWP-induced phosphorylation levels of Akt and eNOS, assessed in cultured endothelial cells from porcine
coronary arteries by Western blot analysis. The present findings indicate that bilitranslocase-mediated
membrane transport substantially contributes to the initial step of RWP-induced coronary vasodilatation.

1 Introduction pathway leading to the activation of endothelial NO synthase

Red wine polyphenols (RWP) have been shown to have multiple
benecial effects on the cardiovascular system, including
improvement of aging-1 and obesity-associated2 endothelial
dysfunction. One of the most studied benecial effects of RWP
is their ability to induce acute endothelium-dependent relaxa-
tions of isolated artery rings,3 related to anti-hypertensive
activity in humans.4 However, one of the major questions
remaining unanswered is how bioactive polyphenols initialize
their signalling pathway in endothelial cells. Plausible expla-
nations include their binding to membrane receptors to
subsequently trigger an intracellular signalling cascade, or their
transport into endothelial cells followed by their binding to
specic molecular targets.
Along with this aspect, in recent years, two main distinct
membrane transport mechanisms for avonoid uptake into
cells have been identied, mediated by bilitranslocase (TC
2.A65.1.1, no SLC available)5,6 and the organic anion-trans-
porting polypeptides 1A2 (TC 2.A.60.1.14, SLCO1A2) and
2B1(TC 2.A.60.1.20, SLCO2B1).7,8 Bilitranslocase is specic for
anthocyanins. Thus, here we hypothesize that the RWP-depen-
dent activation of the redox-sensitive PI3-kinase/Akt-dependent
(eNOS) by phosphorylation at Ser1177 and the subsequent
endothelium-dependent relaxation of porcine coronary artery
rings3,9 may be subordinated to the activity of the avonoid
membrane transporter bilitranslocase.10
To address this question experimentally, we used specic
bilitranslocase antibodies, which bind to an extracellular
domain of the transporter and inhibit its avonoid transport
function.6,11 In the present study, we show the important role of
the bilitranslocase membrane transporter in initiating RWP-
induced intracellular responses of endothelial cells leading to
vasorelaxation.
2 Methods
Bilitranslocase antibodies
The polyclonal bilitranslocase antibodies were obtained from
rabbit sera immunized with a peptide (EDSQGQHLSSF) corre-
sponding to segment 65–75 of the primary structure of bili-
translocase. The antibodies were puried by affinity
chromatography from immune sera of rabbits as previously
described.12,13

Preparation of red wine polyphenols

a
Department of Life Sciences, University of Trieste, via L. Giorgieri 1, 34127 Trieste,
Italy. E-mail: spassamonti@units.it; Fax: +39 0405583691; Tel: +39 0405588747 The RWP dry powder was obtained from French red wine
b
UMR CNRS 7213, Laboratoire de Biophotonique et Pharmacologie, Faculté de (Corbières A.O.C., vintage 2001, composed of a blend of Cari-
Pharmacie, Université de Strasbourg, Illkirch, France gnan, Grenache Noir, and Syrah), provided by Dr M. Moutounet

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Paper
(Institut National de la Recherche Agronomique, Montpellier,
France), and analyzed by Pr. P. L. Teissedre (UFR d'Oenologie,
Université de Bordeaux Segalen, France). The detailed prepa-
ration and analysis of the RWP have been described previ-
ously.14 One liter of red wine produced 2.9 g of RWP, which
contained 471 mg g
1 of total phenolic compounds expressed
as the gallic acid equivalent. The phenolic levels in the used
powder of RWP contained 8.6 mg g
1 of catechin, 8.7 mg g
1 of
epicatechin, dimers (B1: 6.9 mg g
1, B2: 8.0 mg g
1, B3: 20.7 mg
g
1 and B4: 0.7 mg g
1), anthocyanins (malvidin-3-glucoside:
11.7 mg g
1, peonidin-3-glucoside: 0.66 mg g
1, and cyanidin-3-
glucoside: 0.06 mg g
1) and phenolic acids (gallic acid: 5.0 mg
g
1, caffeic acid: 2.5 mg g
1, and caaric acid: 12.5 mg g
1).
Polyphenol-rich juice from Aronia melanocarpa
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To support the observations of red wine polyphenols, we used
another source of polyphenols; namely, the concentrated juice
(66  Bx) from Aronia melanocarpa, also known as chokeberry,
which was provided by Eckes-Granini (Nieder-Olm, Germany).
The original juice was obtained by dilution to 15  Bx in distilled
water. The nal Aronia juice contained 7.15 g L
1 of polyphenols
expressed as the gallic acid equivalent. The major phenolic
compounds present in Aronia melanocarpa are procyanidins,
chlorogenic acids, cyanidin and quercetin glycosylated
derivatives.15
Primary culture of porcine coronary artery endothelial cells
Primary cultures of endothelial cells were prepared as described
previously.16 Briey, le circumex coronary arteries were excised
from pig hearts freshly collected from the local slaughterhouse
(Holtzheim, France). Coronary arteries were cleaned of connective
tissue, ushed with PBS without calcium to remove the remaining
blood, and thereaer exposed to collagenase treatment (type I,
Worthington, 1 mg mL
1 for 12 min at 37  C). Endothelial cells
were cultured in culture dishes containing MCDB 131 medium
(Invitrogen) supplemented with 15% fetal calf serum, penicillin
(100 U mL
1), streptomycin (100 U mL
1), fungizone (250 mg
mL
1), and L-glutamine (2 mM) (all from Cambrex), and grown for
48–72 h. All experiments were performed with sub-conuent
cultures of cells used at the rst passage. Prior to treatment, cells
were exposed to serum-free culture medium in the presence of
0.1% bovine serum albumin (Euromedex) for 6 h.
Western blot analysis
Endothelial cells were incubated with polyclonal bilitranslocase
antibodies (1 mg IgG/mL, in serum-free culture medium) for 30
min before the addition of RWP (50 mg mL
1) or Aronia mela-
nocarpa juice (AMJ, 33.6 mg mL
1 of polyphenols) for 5 min. In
preliminary experiments, the concentration and exposure time
for both treatments were optimized in order to have the best
conditions for studying the activity of transporter proteins, such
as low concentrations of the substrate and a short exposure
time to avoid nonspecic cell membrane transport. Aer
treatment, cells were treated by the protocol previously
described.16 Briey, cells were washed twice with PBS and then
lysed in extraction buffer. The total amount of proteins (20 mg)
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Food & Function
was separated on 10% SDS-polyacrylamide gels, and transferred
electrophoretically to polyvinylidine diuoride membranes
(Amersham). The membranes were blocked for 1 h with Tris-
buffered saline solution with 0.1% Tween-20 (TBS-T) containing
3% bovine serum albumin. For the detection of proteins, the
membranes were incubated overnight at 4  C with the respec-
tive primary antibody (beta-tubulin and eNOS, BD Transduction
Laboratories; p-Akt Ser473 and p-eNOS Ser1177, cell signaling
technology; dilution of 1 : 1000). Aer washing, membranes
were incubated with the corresponding secondary antibody
(HRP-labelled anti-rabbit IgG, dilution of 1 : 5000; HRP-labelled
anti-mouse IgG, dilution of 1 : 20 000; cell signaling technology)
at room temperature for 60 min. The immunoreactive bands
were detected by enhanced chemiluminescence (Amersham).
Densitometric analysis of the bands was performed using
ImageJ soware (NIH, Bethesda, USA).
Vascular reactivity studies
The le circumex coronary arteries from pig heart were cleaned
of connective tissue and cut into rings (4–5 mm in length). The
rings were suspended in organ baths containing oxygenated (95%
O2 and 5% CO2) Krebs bicarbonate solution (composition in mM:
NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.18, CaCl2 1.25, NaHCO3
25, and D-glucose 11, pH 7.4, 37  C) containing the cyclooxygenase
inhibitor indomethacin (10 mM), for the detection of changes in
isometric tension. Following a 90 min equilibration period under
a resting tension of 5 g, the rings were contracted with KCl
(80 mM). Aer a 30 min washout period, the rings were contracted
with the thromboxane mimetic U46619 (1–60 nM) to about 80%
of the maximal contraction before addition of bradykinin (0.3 mM)
to check the presence of a functional endothelium. Only rings
with a functional endothelium (more than 80% relaxation) were
included in the study. Aer the washout and a 30 min equili-
bration period, the rings were again contracted with U46619 to a
plateau level before the construction of a concentration–relaxa-
tion curve to RWP (0.1 mg mL
1 to 0.1 mg mL
1 of total phenolic
compounds expressed as the gallic acid equivalent). To study the
role of bilitranslocase, some coronary artery rings were incubated
for 30 min with anti-sequence polyclonal bilitranslocase anti-
bodies before the addition of U46619 (BTL Ab group), and they
were compared to control rings (CTRL group). Additional controls
with non-specic rabbit IgG were performed as previously
described (not reported here).17 Furthermore, in some experi-
ments, rings were exposed to a pharmacological inhibitor
together with (BTL Ab) or without (CTRL) bilitranslocase anti-
bodies for 30 min before the addition of U46619. The NO-medi-
ated component of the relaxation was determined in the presence
of indomethacin (10 mM) and charybdotoxin plus apamin
(100 nM each) to prevent the formation of vasoactive prostanoids
and EDH-mediated responses, respectively. The EDH-mediated
component was determined in the presence of Nu-nitro-L-arginine
(L-NA, eNOS inhibitor, 300 mM) and indomethacin.
Statistical analyses
All values are expressed as means  SEM. Statistical analyses of
the data were performed using GraphPad Prism version 5.00
Food Funct., 2013, 4, 1452–1456 | 1453

Food & Function
(GraphPad Soware, San Diego, California, USA). Regarding
Western blot experiments, the one-way analysis of variance
(ANOVA) with post Dunnett's test was used to compare the
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Fig. 1 Bilitranslocase antibodies significantly reduce endothelium-dependent
relaxations induced by RWP in porcine coronary artery rings. Coronary artery rings
with endothelium were contracted with U46619 in the absence (C, CTRL) or
presence of bilitranslocase antibodies (B, BTL Ab) before the addition of
increasing concentrations of RWP. Panel (A) RWP-induced relaxations in the
presence of indomethacin (10 mM). Panel (B) RWP-induced NO-mediated relax-
ation. Rings were incubated for 30 min with charybdotoxin + apamin (inhibitors
of endothelium-dependent hyperpolarization, both at 100 nM) and indometh-
acin (inhibitor of cyclooxygenase, 10 mM) to prevent the relaxation mediated by
EHDF and vasoactive prostanoids, respectively, before the contraction with
U46619. Panel (C) RWP-induced EDH-mediated relaxation. Rings were incubated
for 30 min with L-NA (NO synthase inhibitor, 300 mM) and indomethacin (10 mM)
to prevent the formation of NO and vasoactive prostanoids, respectively, before
the contraction with U46619. Results are expressed as means  SEM of 5 different
experiments. n represents the number of coronary artery rings, each obtained
from a different animal. **P < 0.01, ***P < 0.001.
1454 | Food Funct., 2013, 4, 1452–1456
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treated groups to the control group, while in vascular reactivity
studies, Student's t-test was used. Results were considered to be
statistically signicant when P < 0.05.
3 Results and discussion
In a previous study, we observed that avonoids such as bilberry
anthocyanins induced increased coronary ow in the isolated
rat heart under ischemia-reperfusion conditions.18 This effect
was accompanied by an increased post-ischemic coronary blood
ow and le ventricular pressure, and decreased release of
lactate dehydrogenase, indicating a major improvement of
myocardial function. Similar ndings have been obtained using
pure cyanidin 3-glucoside, shown to enter into endothelial cells
via the avonoid transporter bilitranslocase.6
Here, experiments have tested the involvement of bili-
translocase-mediated membrane transport in the vascular
function of porcine coronary arteries. This preparation offers
the opportunity to carry out observations in arterial rings,
which can be suspended in organ baths to assess their vas-
orelaxation in response to pharmacological agents. At the
same time, suitable numbers of endothelial cells can be
explanted and cultured to study the avonoid-dependent
activation of cell signalling pathways, ultimately, leading to
the activation of both NO- and EDH-dependent pathways.
Finally, it should be noted that porcine preparations are
regarded as the animal model nearest to humans, with a high
translational value.
To investigate the specic role of the bilitranslocase-medi-
ated transport of avonoids in initiating vasorelaxation, we
used puried anti-sequence antibodies directed against an
extracellular domain of this membrane transporter, which have
previously been shown to be involved in anthocyanin-induced
relaxations of rat aortic rings,6,17 and also in the uptake of
avonoids into endothelial cells.6,11 In the present study,
experiments were carried out using a RWP standard mixture,
which has previously been shown to induce potent endothe-
lium-dependent relaxations of isolated blood vessels involving
both NO and EDHF.3,9 Clearly, this mixture represents a food
matrix rich in known bioactive compounds that are transported
by bilitranslocase, such as anthocyanins and other
polyphenols.5,19
Bilitranslocase inhibition impairs RWP-induced
endothelium-dependent relaxation of coronary arteries
In porcine coronary artery rings with endothelium, RWP
induced dose-dependent relaxations that are signicantly
impaired by pre-incubation with BTL antibodies (0.24 mg ml
1,
Fig. 1A). The involvement of bilitranslocase was further
assessed on both the NO- (Fig. 1B) and EDH-mediated (Fig. 1C)
relaxations induced by RWP. In both cases, BTL antibodies
signicantly impaired the RWP-induced relaxation of coronary
artery rings (Fig. 1B and C).
Previous studies have also shown that inhibition of bili-
translocase using anti-sequence antibodies inhibited the
anthocyanin-induced relaxation of rat aortic rings, but was
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without effect on the relaxation induced either by acetylcholine,
a physiological activator of NO, or by sodium nitroprusside, a
NO donor.17 Taken together, these data indicate that the RWP-
induced relaxation is dependent, at least in part, on bili-
translocase, most likely due to the transport of polyphenols into
endothelial cells. However, the BLT antibodies only partially
inhibited the RWP-induced relaxation, suggesting the partici-
pation of other cellular uptake mechanisms for polyphenols,
such as via other transporters, or entry by passive membrane
permeation.
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Fig. 2 Bilitranslocase antibodies inhibit RWP- and AMJ-induced phosphorylation
of Akt (Ser473) and eNOS (Ser1177) in endothelial cells. Cultured endothelial cells
were treated with either RWP or Aronia melanocarpa juice (AMJ), with or without
a 30 min pre-incubation period with bilitranslocase antibodies (BTL Ab), before
Western blot analysis of the phosphorylation levels of Akt at Ser473 (Ser473) and
eNOS at Ser1177 (Ser1177). Panel (A) representative immunoblots. Panel (B) and
(C) corresponding cumulative data of the phosphorylation levels of Akt (Panel (B))
and eNOS (Panel (C)) proteins. The results are shown as means  SEM of
5 different experiments, with ns ¼ not significantly different from the control
without BTL Ab; **P < 0.01; *P < 0.05 versus the respective control (CTRL), and
##
P < 0.01; #P < 0.05 versus without BTL Ab.
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Bilitranslocase inhibition prevents RWP-induced
phosphorylation of Akt and eNOS in endothelial cells
Previous studies have reported that RWP activate an intracellular
signalling pathway leading to the activation of eNOS in cultured
coronary artery endothelial cells.3 This stimulatory pathway
involves the redox-sensitive activation of the Src/PI3-kinase
pathway leading to the phosphorylation of Akt at serine 473,
which, in turn, activates eNOS by phosphorylation of serine 1177.3
In the present study, exposure of cultured endothelial cells to
either RWP or the polyphenol-rich Aronia melanocarpa juice
(AMJ) for 5 min signicantly increased the phosphorylation
levels of both Akt (Ser473) and eNOS (Ser1177) (Fig. 2). AMJ was
included because it is rich in anthocyanins and thus served as
an additional test. We speculated that the membrane transport
of RWP and AMJ avonoids into endothelial cells occurs via
bilitranslocase, and hence, that the inhibition of bilitranslocase
activity reduces phosphorylation of the aforementioned protein
targets. Indeed, a signicant reduction of both RWP- and AMJ-
induced phosphorylation levels of Akt and eNOS was observed
following pre-incubation of cells with BTL antibodies (Fig. 2). In
contrast, pre-incubation of endothelial cells with rabbit IgG
immunoglobulins did not have such an effect (data not shown).
Moreover, incubation of endothelial cells with bilitranslocase
antibodies per se did not affect the low phosphorylation level of
either Akt or eNOS (Fig. 2, control lanes, ns marked).
Our ndings support the view that the bilitranslocase-
dependent transport of RWP operates as an early mechanism in
mediating the endothelium-dependent relaxation of coronary
arteries in response to polyphenols, as summarised in Fig. 3.
These data ll, at least in part, the gap of knowledge in
explaining how bioactive polyphenols occurring in red wine
initialize their signalling pathway in endothelial cells. However,
it can be speculated that this membrane carrier might not be
the unique player in this scenario and other membrane trans-
porters might contribute to this complex event. Therefore,
additional investigations are crucially warranted to further
characterize the early events involved in the interaction of
Fig. 3 Bilitranslocase-dependent transport of anthocyanins operates as an early
mechanism in mediating the endothelium-dependent relaxation of coronary
arteries in response to red wine polyphenols.
Food Funct., 2013, 4, 1452–1456 | 1455

Food & Function
polyphenols with endothelial cells. In addition, the involvement
of other signalling pathways, including passive entry into the
cells or activation of membrane receptors, should be consid-
ered. Indeed, the role of estrogen receptor alpha in the relaxa-
tion induced by a red wine phenolic extract has been reported in
mouse aorta.20 However, this role remains controversial as other
studies indicated that the RWP-induced relaxation is indepen-
dent of estrogen receptor alpha in rat aorta and porcine coro-
nary artery.21,22 Moreover, in the present study, the pre-
incubation of porcine coronary artery rings with fulvestrant, a
specic estrogen receptor alpha antagonist, did not inhibit the
RWP-induced relaxation (data not shown).
Since the present study used porcine coronary artery rings and
cells, further studies are warranted before one can extrapolate the
present ndings to humans. However, the presence of the bili-
translocase transporter in the membrane of commercial primary
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endothelial cells explanted from human aorta has been previ-
ously described.11 It can be speculated that the bilitranslocase-
mediated transport of RWP in human endothelial cells might
lead to the formation of the potent vasoprotective factor NO.
In conclusion, the present study provides the rst evidence
that the activation of cell signalling pathways leading to vaso-
dilation is subordinate, at least in part, to the trans-membrane
passage of red wine bioactive compounds from the medium
into the vascular endothelium.
Conflicts of interest
None declared.
Abbreviations
ARJ Aronia juice;
BTL Bilitranslocase;
eNOS Endothelial nitric oxide synthase;
RWP Red wine polyphenols.
Acknowledgements
Research was supported, in part, by the Office National Inter-
professionnel des Fruits, des Légumes, des Vins et de l'Horti-
culture (Action Vin & Santé, France), by the Slovenian Research
Agency for the young research fellowship awarded to L. Z.
(411476–1/2007), and by the European Regional Development
Fund, the Cross-Border Cooperation Italy-Slovenia Programme,
2007–2013 (strategic project TRANS2CARE to S.P.).
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