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The FASEB Journal • Research Communication
Effects of red wine polyphenols on postischemic
neovascularization model in rats: low doses are
proangiogenic, high doses anti-angiogenic
Celine Baron-Menguy,* Arnaud Bocquet,* Anne-Laure Guihot,* Daniel Chappard,
†
Marie-Joseph Amiot,
‡
Ramaroson Andriantsitohaina,* Laurent Loufrani,*
and Daniel Henrion*
,1
*CNRS UMR 6214, Angers, France; INSERM UMR 771, Angers, France; Universite´ d’Angers, UFR de
Me´decine, France;
†
INSERM, EMI 0335, Angers, France; and
‡
UMR 476 INSERM/1260 INRA,
Marseille, France
ABSTRACT Polyphenols, present in green tea, grapes,
or red wine, have paradoxical properties: they protect
against cardiac and cerebral ischemia but inhibit angio-
genesis in vitro. So we investigated the effects of polyphe-
nols in vivo on postischemic neovascularization. Rats
treated with low (0.2 mg 䡠 kg
ⴚ1
䡠 day
ⴚ1
) or high (20 mg 䡠
kg
ⴚ1
䡠 day
ⴚ1
) doses of red wine polyphenolic compounds
(RWPC) were submitted to femoral artery ligature on the
left leg. Two wks after ligature, high doses of RWPC (i.e.,
7 glasses of red wine) reduced arterial, arteriolar, and
capillary densities and blood flow in association with an
inhibition of a PI3 kinase-Akt-endothelial NO synthase
(eNOS) pathway, decreased VEGF expression, and lower
metalloproteinase (MMP) activation. Low doses of RWPC
(i.e., 1/10th glass of red wine) increased the left/right
(L/R) leg ratio to control level in association with an
increased blood flow and microvascular density. This
angiogenic effect was associated with an overexpression
of PI3 kinase-Akt-eNOS pathway and an increased VEGF
production without effect on MMP activation. Thus, low
and high doses RWPC have respectively pro- and anti-
angiogenic properties on postischemic neovascularization
in vivo. This unique dual effect of RWPC offers important
perspectives for the treatment and prevention of ischemic
diseases (low dose) or cancer growth (high dose).—
Baron-Menguy, C., Bocquet, A., Guihot, A.-L., Chappard,
D., Amiot, M.-J., Andriantsitohaina, R., Loufrani, L., Hen-
rion, D. Effects of red wine polyphenols on postischemic
neovascularization model in rats: low doses are proangio-
genic, high doses anti-angiogenic. FASEB J. 21, 3511–3521
(2007)
Key Words: VEGF 䡠 remodeling 䡠 nitric oxide 䡠 blood flow
Angiogenesis is a process involved in several physi-
ological events including embryonic development, fe-
male reproductive cycle placentation, and wound re-
pair. It also takes part in various pathological
conditions such as tumor growth, diabetic retinopathy,
rheumatoid arthritis (1, 2), and ischemic disease. An-
giogenesis is a very complex process involving a variety
of biologically active substances (3).
Epidemiological studies report an inverse association
between polyphenol consumption such as fruits and
vegetables, tea, and red wine and mortality from car-
diovascular diseases and cancers. Polyphenols, particu-
larly red wine polyphenols (RWPC), exert numerous
effects including antioxidant and free radical proper-
ties, and anti-aggregatory platelet and anti-thrombotic
activities. With regard to blood vessels, RWPC are
powerful vasodilators and contribute to preserving the
integrity of the endothelium, inhibition of vascular cell
proliferation and migration, including endothelial and
smooth muscle cells, and angiogenesis (4, 5).
Concerning angiogenesis, RWPC exert paradoxical
properties. On the one hand, RWPC protect against
deleterious effect of cardiac and cerebral ischemia
whose correction requires proangiogenic properties to
produce new blood vessels to rescue the infracted area,
both in human and different experimental models
(6–8). In addition to the antioxidant properties of
RWPC, the mechanisms involved mainly the activation
of endothelial NO release through an increase in
calcium level and activation of the PI3-kinase/Akt
pathway by a redox-sensitive pathway in endothelial
cells (9–11). RWPC may also regulate NO activity at the
level of endothelial NO synthase (eNOS) protein ex-
pression in endothelial cells (12), blood vessels, and
cardiac tissue (13). On the other hand, numerous
studies report that RWPC inhibit angiogenesis by acting
on different vascular cells. Indeed, RWPC or its con-
tent, delphinidin, inhibit endothelial cell proliferation
through the involvement of cyclin D1- and A-depen-
dent pathways (14, 15). Moreover, RWPC decrease
vascular smooth muscle migration and proliferation
through down-regulation of cyclin A expression and
inhibition of p38 MAPK and PI3-kinase pathways (16).
RWPC was recently shown to inhibit expression of the
1
Correspondence: Department of Neurovascular Inte
-
grated Biology, UMR CNRS6214-INSERM771, Faculte´de
Me´decine, 49045 Angers, France. E-mail: daniel.henrion@
univ-angers.fr
doi: 10.1096/fj.06-7782com
35110892-6638/07/0021-3511 © FASEB
two major proangiogenic factors, vascular endothelial
growth factor (VEGF) (17) and metalloproteinase-2
(MMP-2) (18), in smooth muscle cells. All of these
effects confer both in vitro and in vivo anti-angiogenic
properties of RWPC and its polyphenols content (14,
17).
The mechanisms by which RWPC exert these para-
doxical effects on angiogenesis are not understood. In
the present study we tested the hypothesis that RWPC,
used chronically, might exert a dual effect depending
on the dose used. Formation of new blood vessels in
chick chorioallantoic membrane (CAM) has been used
as a model to test the anti-angiogenic properties of
RWPC. However, it is important to consider a valuable
in vivo model that takes into account ischemia, the
driving force for angiogenesis, activation of NO and
hypoxia-inducible factor 1␣ (HIF1␣), and the produc-
tion of VEGF. Therefore, we used a model of femoral
artery ligature leading to a 50% reduction in blood
vessel density in the ischemic leg musculature, which
allowed the characterization of pro- and anti-angio-
genic effects of RWPC.
MATERIALS AND METHODS
Animals
Protocol 1: Effects of Provinols™ in angiogenesis
Twelve-wk-old male normotensive Wistar rats (IFFA-CREDO,
l’Arbresle, France) were treated with a high dose (HD, 20
mg 䡠 kg
⫺1
䡠 day
⫺1
, n⫽9), an intermediate dose (2
mg 䡠 kg
⫺1
䡠 day
⫺1
, n⫽5), and a low dose (LD 0.2
mg 䡠 kg
⫺1
䡠 day
⫺1
, n⫽9) of RWPC by daily gavage for 21 days.
Provinols™ represents the polyphenolic compounds isolated
from red wine and involves (in mg/g of dry powder) 480
proanthocyanidins, 61 total anthocyanins, 19 free anthocyanins,
38 catechin, 18 hydroxycinnamic acids, 14 flavonols, and 370
polymeric tannins (13). RWPC were dissolved in a solution of
5% glucose (Glc). Consequently, the control group received
only 5% glucose (n⫽12) in the same conditions.
Protocol 2: Involvement of VEGF pathway
To assess the involvement of VEGF, four additional groups
were performed: a control group that received 5% glucose by
daily gavage (n⫽5), a control group treated with VEGF
neutralizing antibody (2.5 g, i.p. twice a wk, R&D Systems,
Minneapolis, MN, USA; n⫽5), a group treated with a low dose
of RWPC (n⫽5), and one with or without VEGF neutralizing
antibody (19).
Protocol 3: Effects of delphinidin in angiogenesis
Delphinidin, a compound of RWPC, was also tested to
identify, in vivo, its involvement in the angiogenic process.
Consequently, two groups, treated with a high dose (0.6
mg 䡠 kg
⫺1
䡠 day
⫺1
, n⫽6) and a low dose (0.06
mg 䡠 kg
⫺1
䡠 day
⫺1
, n⫽6) of delphinidin were performed by
daily gavage for 15 days. The control group received only 5%
glucose (n⫽5) in the same conditions.
Animals were housed in a regulated environment with a
constant ambient temperature of 24°C. They had free access
to standard laboratory food and water. As described earlier
(19), after 7 days of treatment the femoral artery was oc-
cluded (3-0 silk suture) under anesthesia (Nesdonal, 50
mg/kg, i.p.). The ligature was performed on the left femoral
artery 0.5 cm proximal to the bifurcation to the saphenous
and popliteal arteries. After 7 (protocols 2, 3) or 15 (protocol
1) days of ligature, blood flow was measured as described
below, followed by an angiographic measurement. The rat
was then sacrificed and tissues were sampled for biochemical
and histological analysis. The procedure followed in the care
and euthanasia of the study animals was in accordance with
the European community standards on the care and use of
laboratory animals (authorization #00577).
Laser-Doppler whole body imaging
To provide a functional evidence of ischemia, after 15 days of
ligature laser Doppler perfusion was performed in anesthe-
tized rats. Animals were settled in an incubator (MMS,
Chelles, France) that allowed maintenance of a stable cuta-
neous temperature (35.0⫾0.5°C) throughout the experi-
ment. Perfusion was then measured in the foot using a laser
Doppler flow probe (PF 408; Perimed). Blood flow was
recorded for 5 min. At least two flow measurements were
performed per leg. Blood flow perfusion was expressed as a
ratio of left (ischemic) to right (nonischemic) leg.
To evaluate the neovascularization (protocol 2), laser
Doppler perfusion imaging experiments were performed on
day 7 as described (19). Briefly, rats were placed on a heating
plate at 37°C to minimize temperature variation. To account
for variables, including ambient light, temperature, and ex-
perimental procedures, blood flow was calculated in the foot
and expressed as a ratio of ischemic to nonischemic leg.
Angiography
Arterial density was evaluated by high-definition angiography
1 or 2 wk after ligature, as described (19). Briefly, rats were
anesthetized (sodium pentobarbital, 50 mg/kg, i.p.) and a
contrast medium (barium sulfate, 6g/ml) was injected
through a catheter introduced into the abdominal aorta. Two
images were acquired per animal using a digital X-ray trans-
ducer (Faxitron X-Ray Corporation, Wheeling, IL, USA).
Vascular density was expressed as a percentage of pixels per
image occupied by vessels in the quantification area. The
quantification zone was delineated by the location of the
ligature on the femoral artery, the knee, the edge of the
femur, and the external limit of the leg. The angiographic
score was calculated as the ratio of left (ischemic) to right
(nonischemic) leg (L/R ratio). In each experiment, the
skeletal muscle from both the ischemic and nonischemic leg
were removed and frozen before Western blot and histologi-
cal analysis, as described before (19).
Arteriolar and capillary density
Angiographic analysis was completed by assessing capillary
and arteriolar densities, as described (20). Briefly, ischemic
and nonischemic muscles were dissected and progressively
frozen in isopentane solution cooled in liquid nitrogen.
Sections (7 m) were first incubated for 30 min in PBS
containing 5% BSA at room temperature, then for 1 h with
either mouse monoclonal antibody directed against human
smooth muscle actin ␣
1
(dilution 1:50) to identify arterioles
or rabbit polyclonal antibody directed against total PECAM
(dilution 1:50) to identify capillaries. Capillary-to-myocyte
and arteriole-to-myocyte ratios were calculated, and the re-
sults were expressed according to ischemic-to-nonischemic
3512 Vol. 21 November 2007 BARON-MENGUY ET AL.The FASEB Journal
ratios. Capillary and arteriolar densities were calculated in
three randomly chosen fields of a definite area for each
animal.
Western blot in isolated muscles
In each experiment, skeletal muscle from both ischemic and
nonischemic leg was removed and frozen. Samples were
collected and homogenized (Polytron Pro 250, Bioblock
Scientific, Illkirch, France). Proteins were separated by SDS-
PAGE (Mini gel protean II system, Bio-Rad (Hercules, CA,
USA), 100V, using 300 ml 25 mM Tris, 192 mM glycine, 0.1%
SDS) using a stacking gel, followed by a running gel. After
migration, proteins were transferred (100 V, 2 h, 4°C using
800 ml 25 mM Tris, 192 mM glycine, 10% methanol) to PVDF
blotting membranes (Immobilon-P, Millipore, Billerica, MA,
USA). Membranes were then washed in TBS-T buffer (com-
position: 10 mM Tris/base pH 7.5, 0.1 M NaCl, 1 mM EDTA,
0.1% Tween 20) and blocked for1hatroom temperature
(5% BSA in TBS-T). Membranes were incubated overnight
4°C with the primary antibody (1:500), washed again (3 times
for 10 min), then incubated with HRP-conjugated secondary
antibody (Amersham; Arlington Heights, IL, USA; 1 h, 30
min RT, 1:2000). Membranes were washed and visualized
using the ECL-Plus Chemiluminescence kit (Amersham).
Immunodetection was carried out using antibodies directed
against Akt, P-Akt, eNOS, P-eNOS, VEGF, caveolin-1, HSP 90,
and RhoA (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Gelatin zymography
Tissue samples were thawed and homogenized in 300 lof
buffer (200 mmol/L sucrose and 20 mmol/L HEPES, pH 7.4)
containing protease inhibitors. Protein content was then
determined by the method of Bradford (21). Samples were
mixed in an SDS-PAGE loading buffer (lacking reducing
agents) applied to SDS/9% polyacrylamide gels containing 1
mg/ml gelatin (Bio-Rad) and separated by electrophoresis.
Subsequently, SDS was removed from the gels by two 15 min
washes with 2.5% Triton X-100, then the gels were incubated
overnight at 37°C in zymography buffer (50 mmol/L Tris
[pH 7.5] and 10 mmol/L CaCl
2
) and stained with Coomassie
Brilliant Blue (Serva, Heidelberg, Germany). Gelatinolytic
activity was visualized as clear areas of lysis in the gel.
Densitometric analysis was performed using NIH Image soft-
ware.
Data analysis
Results are expressed as means ⫾ se. Significance of the
differences between groups was determined by analysis of
variance (ANOVA), followed by Bonferroni’s test and paired
t test. P values of ⬍ 0.05 were considered significant.
RESULTS
Animals and blood pressure after Provinols™
treatment
Before the experimental protocol, body weight was
equivalent in different groups of rats. After 21 days of
treatment with a high dose of RWPC, body weight of
the rat was significantly lower than in the control group
(310.0⫾7.2 vs. 333.6⫾5.9 g, P⬍0.05). Treatment with
intermediate and low doses of RWPC did not affect
body weight. Blood pressure was significantly decreased
by a high dose of RWPC (103.4⫾0.9 vs. 107.3⫾1.2
mmHg, P⬍0.05, n⫽9). A low dose of RWPC (n⫽7) had
no significant effect on blood pressure (data not
shown).
Quantification of neovascularization in the hind limb
Protocol 1: Whole body laser Doppler blood flow
Fifteen days after ligature, foot blood flow as measured
using laser Doppler perfusion imaging was significantly
lower in the left leg than in the right leg, with an L/R
ratio of 76.7 ⫾ 7.4%. A high dose of RWPC decreased
by 0.40-fold the L/R ratio (30.9⫾6.6, P⬍0.01) com-
pared with control, whereas a low dose increased by
1.48-fold the L/R ratio (114⫾12.5, P⬍0.05) compared
with control. The intermediate dose of RWPC did not
affect the angiogenic process (Fig. 1A).
Angiography
Fifteen days after ligature, the microvascular network
density was lower in the left leg than in the right leg,
with an L/R ratio of 67.5 ⫾ 4.5 (P⬍0.05) in the control
group (Fig. 1B). Treatment with a high dose of RWPC
significantly decreased the L/R ratio to 38.4⫾2.5
(P⬍0.05) compared with control. On the other hand, a
low dose of RWPC increased the L/R ratio up to
119.5 ⫾ 15.8 (P⬍0.001) compared with the control
group, whereas 2 mg/kg of RWPC did not affect the
vascular density (Fig. 1B).
Arteriolar and capillary densities
The angiogenic process was not affected by the inter-
mediate dose of Provinols™. Consequently, we concen-
trated our work on the two others doses (0.2 and 20
mg 䡠 kg
⫺1
䡠 day
⫺1
). Data from the angiographic analysis
were confirmed by arteriolar density measurement.
Fifteen days after ligature, the L/R ratio was 85 ⫾ 6%,
with an arteriolar density in the left leg increased by
0.94-fold for the control group. A concurrent treatment
with a high dose of RWPC decreased by 0.62-fold the
L/R ratio (P⬍0.05) compared with controls, indicating
an anti-angiogenic effect (Fig. 2A). A low dose of RWPC
increased by 1.59-fold the L/R ratio (P⬍0.01) com-
pared with control, indicating a proangiogenic effect
(Fig. 2A).
In the control group, capillary density was not af-
fected by the ligature. A high dose of RWPC induced a
decrease of L/R ratio by 0.77-fold (P⬍0.05) compared
with the control group (Fig. 2B). No difference was
observed with RWPC low-dose treatment compared
with controls.
Western blot analysis
Angiogenesis in the control group (Fig. 3)
In the control group, the expression level of eNOS,
phosphorylated-eNOS, and HSP 90 in the ischemic (L)
3513RED WINE POLYPHENOLS, DUAL EFFECTS ON ANGIOGENESIS
hind limb was significantly increased compared with
nonischemic (R) leg (n⫽6, P⬍0.05), as described in
the literature. No change with caveolin-1 expression
was observed. The ischemia induced a rise of Akt
pathway, with an increase of Akt (P⬍0.05), P-Akt
(P⬍0.05), and PI3K (P⬍0.05) levels in the ligatured
hind limb. p38 and P-p38 were not significantly modi-
fied in the left leg (P⬍0.05). Finally, VEGF and NF-B
expressions were increased in the ischemic leg com-
pared with the nonischemic leg (P⬍0.05).
Figure 1. Quantitative evaluation of legs neovascularization 15 days after femoral artery ligature in rats treated with 0.2, 2, and
20 mg 䡠 kg
⫺1
䡠 day
⫺1
of polyphenols (RWPC) for 21 days. Blood flow (n⫽6/group) with typical images and vascular density with
typical radiograms (n⫽9/group) are respectively shown in panels A, B. Values are expressed in mean ⫾ se as the
ischemic/nonischemic leg ratio. **P ⬍ 0.01, ***P ⬍ 0.001 vs. control group.
Figure 2. Evaluation of arteriolar (A) and capillary (B) densities in rats treated with 0.2 and 20 mg/kg for 21 days. Values are
expressed in mean ⫾ se as the ischemic/nonischemic leg ratio. *P ⬍ 0.05, **P ⬍ 0.01 vs. control group.
3514 Vol. 21 November 2007 BARON-MENGUY ET AL.The FASEB Journal
Effect of RWPC in the aorta (Fig. 4)
Compared with control, a high dose of RWPC in-
creased expression of eNOS, P-eNOS, and HSP90.
This dose also induced an increase of Akt and PI3K
expression without affecting P-Akt. Finally, a high
dose of RWPC did not affect p38, P-p38, or VEGF
expression.
Compared with control, a low dose of RWPC in-
creased significantly the expression of NO, Akt/PI3K
pathways but decreased that of HSP90. A low dose of
RWPC reduced p38 slightly but markedly increased
P-p38 expression. Finally, a low dose of RWPC did not
affect VEGF expression.
A comparison between the effects of high and low
doses of RWPC indicated a greater activation of NO
pathway as shown by the difference in the ratio of
P-eNOS/eNOS, HSP90 expression, and showed a lower
activation of Akt/P-Akt and P-p38 for the former com-
pared with the latter. The two doses of RWPC increased
the PI3K level equally and neither affected VEGF
expression.
Figure 3. Quantitative evaluation of the expression level of eNOS,
P-eNOS, Akt, P-Akt, PI3K, VEGF, HSP90, caveolin-1, p38, P-p38, and
NF-B, extracted from the right (nonischemic) and left (ischemic)
leg muscle in the control group. Results are expressed in % of the
right leg. Values are mean ⫾ se, n ⫽ 6 per group. *P ⬍ 0.05 and
**P ⬍ 0.01, right (R) vs. left (L) leg.
Figure 4. Quantitative evaluation of the expression
level of eNOS, P-eNOS, Akt, P-Akt, PI3K, VEGF,
HSP90, p38, P-p38, and NF-B in the aorta isolated
from rats treated with 5% glucose (Glc), polyphenols
20 mg/kg (HD), or polyphenols 0.2 mg/kg (LD).
Values are mean ⫾ se, n ⫽ 6 per group. *P ⬍ 0.05,
**P ⬍ 0.01, and ***P ⬍ 0.001 vs. control group.
3515RED WINE POLYPHENOLS, DUAL EFFECTS ON ANGIOGENESIS
Effect of RWPC in the nonischemic hind limb (Fig. 5)
A high dose of RWPC reduced eNOS but markedly
increased P-eNOS expression, and thus enhanced the
ratio P-eNOS/eNOS. HSP90 and caveolin-1 expression
was also increased. A high dose of RWPC did not affect
Akt, P-Akt levels, but significantly increased that of
PI3K. A high dose of RWPC enhanced the p38 MAPK
pathway through an increase in P-p38. Finally, it did not
change VEGF expression and reduced that of NF-B.
A low dose of RWPC increased eNOS but not P-eNOS
expression without a significant change in the P-eNOS/
eNOS ratio. This dose did not affect HSP90 or caveo-
lin-1 expression. A low dose of RWPC significantly
increased Akt expression but did not change that of
P-Akt and PI3K. This dose increased the expression of
both p38 and P-p38. Finally, a low dose did not affect
VEGF expression and significantly enhanced NF-B.
A comparison between the effects of high and very
low doses of RWPC showed a greater activation of NO
pathway, HSP90, caveolin-1, and PI3K expression for
the former compared with the latter. No difference was
observed between the two doses of RWPC with regard
to P-Akt, p38, or the increase of P-p38. A high dose of
RWPC did not affect the Akt level whereas a low dose of
RWPC increased its expression. RWPC induced a dif-
ferential effect on NF-B in which a high dose reduced
but a low dose enhanced its expression. The two doses
of RWPC did not affect VEGF expression.
Effect of RWPC on angiogenesis (Fig. 6)
A high dose of RWPC significantly reduced the expres-
sion of proteins of the NO and Akt/PI3K pathways as
well as HSP90 and caveolin-1 expression. A high dose of
RWPC increased p38 without affecting P-p38 expres-
sion. This dose reduced both VEGF and NF-B expres-
sion.
On the other hand, a low dose of RWPC did not
modify eNOS but increased P-eNOS expression without
any significant change in the ratio P-eNOS/eNOS. This
dose did not affect HSP90 and reduced caveolin-1
expression. A low dose of RWPC did not change the
expression of Akt but significantly increased that of
P-Akt and PI3K. This dose of RWPC increased expres-
sion of both p38 and P-p38. Finally, a low dose of RWPC
significantly enhanced VEGF expression without affect-
ing that of NF-B.
A comparison between the effects of high and low
doses of RWPC showed a lower activation of NO and
Akt/PI3K pathways, HSP90 and P-p38 expression for
the former compared with the latter. The two doses of
RWPC decreased caveolin-1 expression. RWPC induced
a differential effect on VEGF in which a high dose
reduced and a low dose enhanced its expression.
Finally, a high dose reduced NF-B expression but a
low dose did not affect its level.
MMPs and angiogenesis (Fig. 7)
In the control group, MMP activity was significantly
increased in the ischemic hind limb compared with the
nonischemic hind limb (⫻1.31), with an L/R ratio of
1.34 ⫾ 0.11 (P⬍0.01).
After 15 days of ligature, a high dose of RWPC
reduced vascular density and blood flow (Fig. 1A, B);
this anti-angiogenic effect was associated with a re-
duced L/R ratio of MMP activity in the ischemic hind
limb (Fig. 7). However, MMP activity did not differ
from a low dose of RWPC compared with control (L/R
ratio: 1.25⫾0.11).
In this study we have demonstrated that a low dose of
RWPC induced a proangiogenic effect by up-regulation
of VEGF/eNOS pathway. Consequently, we have used a
neutralizing VEGF antibody to prove the involvement
of VEGF.
Figure 5. Quantitative evaluation of the expression level of
eNOS, P-eNOS, Akt, P-Akt, PI3K, VEGF, HSP90, caveolin-1,
p38, P-p38, and NF-B in the right (R, nonischemic) leg. The
effect of high (HD) and low doses (LD) of polyphenols
on right leg protein levels was observed after 21 days of
treatment. Results are expressed as percentage of the control
right leg (R Glc). Values are mean ⫾ se, n ⫽ 6 per group. *P ⬍
0.05, **P ⬍ 0.01, and ***P ⬍ 0.001 vs. control group (R Glc).
3516 Vol. 21 November 2007 BARON-MENGUY ET AL.The FASEB Journal
Protocol 2: Involvement of VEGF pathway (Fig. 8)
The changes in vascularization of foot blood flow were
measured after 7 days of ligature. In the control group,
vascularization of the ischemic foot reached 44.3 ⫾
3.1% compared with the nonischemic foot blood flow.
The injection of neutralizing VEGF antibody twice/wk
significantly decreased the L/R ratio by 0.76-fold
(P⬍0.05). Treatment with 0.2 mg/kg of RWPC in-
creased significantly the L/R ratio by up to 54.8 ⫾ 2.8%
compared with controls. The neutralizing VEGF anti-
body treatment decreased by 0.77-fold the L/R ratio
(P⬍0.05) (Fig. 8A).
The quantification of vascular density showed that
the neutralizing VEGF antibody decreased significantly
the L/R ratio by 0.69- and 0.56-fold in the control and
RWPC-treated groups, respectively (P⬍0.05) (Fig. 8B).
Protocol 3: Effects of delphinidin in angiogenesis (Fig. 9)
The effects of delphinidin in the changes in foot
vascularization were measured by laser Doppler flow-
metry and microangiography. Results showed that the
L/R ratio of foot blood flow and vascular density were
significantly decreased after a high dose of delphinidin
(⫻0.76- and ⫻0.67-fold, respectively, P⬍0.05) com-
pared with the control group, but a low dose did not
change vascular density (Fig. 9).
DISCUSSION
Polyphenols in general and RWPC in particular are
reported to possess anti-angiogenic properties. On the
other hand, they protect against deleterious effects of
cardiac and cerebral ischemia, the correction of which
requires proangiogenic properties to produce new
blood vessels in order to rescue the infracted area. This
dual effect of RWPC remains unexplained. The present
study highlights a dose-dependent effect of polyphe-
nols in an in vivo model of angiogenesis triggered by
ischemia. Thus, a high dose of RWPC had anti-angio-
genic properties, whereas at low doses RWPC promote
angiogenesis. A high dose of RWPC reduced angiogen-
esis via an inhibition of NO/VEGF and Akt/PI3K
pathways. MMP activity was reduced in association with
Figure 6. Involvement of proteins in ischemic/nonischemic
angiographic ratio expressed in percent of control ratio. Quan-
titative evaluation of levels expression of eNOS, P-eNOS, Akt,
P-Akt, PI3K, VEGF, HSP90, caveolin-1, p38, P-p38, and NF-B
proteins extracted from leg muscle was performed. Values are
mean ⫾ se, n ⫽ 6 per group. *P ⬍ 0.05, **P ⬍ 0.01, and ***P ⬍
0.001 vs. control group.
Figure 7. Bottom: Representative gelatin zymographic analy-
sis of protein extract from nonischemic (Non isch) and
ischemic (Isch) legs of control rats and treated with a high
and a low dose of RWPC. Gelatin zymographic analysis
revealed a lytic band at 62 kDa corresponding to the active
form of MMP-2, a minor lytic band of 72 kDa consistent with
the pro-form of MMP-2. For each group, 50 g of total
proteins was used. Top: Densitometry analysis of zymography
gels expressed as percentage of control (isch/non isch ratio).
Values are mean ⫾ se, n ⫽ 6 per group. *P ⬍ 0.05 vs. control
group.
3517RED WINE POLYPHENOLS, DUAL EFFECTS ON ANGIOGENESIS
P-p38 and NF-B expression. The data also highlighted
the mechanism by which a low dose of RWPC promotes
angiogenesis and this includes activation of NO, Akt/
PI3K, p38 MAPK, and VEGF expression without alter-
ing either MMP activity or NF-B expression. Thus,
NO/VEGF, MMPs, and NF-B are crucial to determine
the effect of RWPC on angiogenesis.
The present study provides evidence that in vivo
administration of RWPC induced differential effects on
angiogenesis depending on the dose used. In general,
in vivo effects of RWPC have been conducted at doses
ranging between 20 and 40 mg/kg for 1 to 4 wk
corresponding to a high dose of RWPC used in the
present study. A previous study showed that a high dose
of RWPC induces cardiovascular effects, including im-
provement of endothelial function (22) and preventing
the increase in blood pressure in NO-deficient hyper-
tensive rats (23). Also, a high dose of RWPC induces
hypotension, decreases cardiac reactivity, infarct size
(6), and stroke (8) in rats. Correction of cardiac and
cerebral ischemia required proangiogenic properties to
produce new blood vessels and rescue the infracted
area. In contrast to previous studies, the proangiogenic
properties occurred at a dose (0.2 mg/kg) of RWPC
that is 100-fold lower. The molecular identity of the
compounds responsible for the in vivo effects described
in the present study has not been assessed. Neverthe-
less, it was found that low and high doses of RWPC are
adequate to produce a sufficient circulating concentra-
tion of compounds able to modulate angiogenesis. A
balance between circulating pro- and anti-angiogenic
compounds might occur in vivo, and this equilibrium is
shifted toward proangiogenic substances at a low dose
of RWPC or in tissues with low rate of polyphenols
Figure 8. Effects of neutralizing VEGF after 7 days of femoral ligature in controls and in rats
treated with 0.2 mg/kg of RWPC (n⫽6/group) for 15 days. Values are expressed as L/R ratio, vs.
control group. *P ⬍ 0.05 vs. control group and #P ⬍ 0.05 vs. rats treated with 0.2 mg/kg of RWPC.
Figure 9. Effects of delphinidin in blood flow (A) and vascular density (B) after
7 days of ligature in rats treated with 0.6 and 0.06 mg䡠 kg
⫺1
䡠 day
⫺1
of delphinidin
for 15 days (n⫽6/group). Values are expressed in mean se with *P ⬍ 0.05
compared with controls.
3518 Vol. 21 November 2007 BARON-MENGUY ET AL.The FASEB Journal
distribution at a high dose of RWPC. After treatment of
rats with a high dose of delphinidin, one the major
compounds found in RWPC, angiogenesis was signifi-
cantly inhibited. Consequently, the anti-angiogenic ef-
fect of polyphenols was induced, at least in part, by
delphinidin. Nevertheless, a low dose of delphinidin
had no effect on angiogenesis, suggesting that another
compound or the combination of delphinidin and
other related compound might be involved in the
proangiogenic effect of Provinols™.
We used an in vivo model, which is appropriate to
study ischemic angiogenesis, a classical feature of pe-
ripheral, cardiac and cerebral ischemic diseases (24).
This model also allowed the study of both pro- and
anti-angiogenic effects of RWPC under the same con-
ditions. In control rats, we found that angiogenesis
after ligature was associated with increased eNOS, Akt,
VEGF and HSP 90 expression, in agreement with
previous studies conducted in the same model (25, 26).
The two doses of RWPC affected neither angiogene-
sis nor blood flow in the nonischemic area. It is possible
that ischemic conditions are needed for RWPC in vivo
to modulate angiogenesis, and such conditions are not
fulfilled in the nonischemic area. Nevertheless, in the
nonischemic area a high dose of RWPC activates mul-
tiple pathways reported in previous studies including
increase of the Akt/PI3K and NO pathway. A low dose
of RWPC produced a lower activation of the NO
pathway than a high dose of RWPC. Indeed, RWPC has
been shown to activate the PI3-kinase/Akt pathway
leading to rapid and sustained activation and enhanced
expression of eNOS in endothelial cells (27) and in
different blood vessels (27, 28). In the nonischemic leg,
VEGF expression was not altered by the two doses of
RWPC. The increase of VEGF gene expression occurs
via the NADPH oxidase-dependent formation of reac-
tive oxygen species with subsequent activation of redox-
sensitive kinase such as p38 MAPK, resulting in turn in
the activation of the transcription factor HIF1␣ (29,
30). HIF1␣ might not be activated under nonischemic
conditions, although the two doses of RWPC enhanced
p38 MAPK activation. In the nonischemic leg, MMP
activity was either reduced or unchanged by high and
low doses of RWPC, respectively. In accordance with
our studies, RWPC has been reported to prevent throm-
bin-induced activation of MMP-2 in vascular smooth
muscles (18). Finally, in the nonischemic leg we found
that a high dose of RWPC reduced whereas a low dose
increased NF-B expression. This differential effect of
RWPC has never been reported, but activation of NF-B
can lead to the production of secreted factors that
enhance growth, survival, and vascularization; this in
accordance with the proangiogenic property of RWPC
reported in the present study (see below).
A major finding was the effect of RWPC under
ischemic conditions. In the ischemic leg, we found
that a high dose of RWPC reduced angiogenesis in
association with decreased blood flow and a lower
arteriolar and capillary density via an inhibition of
the NO and Akt/PI3K pathways. However, the mech-
anism by which a high dose of RWPC reduced the NO
pathway under ischemic conditions is not known; this
dose of RWPC increases NO expression and activity.
We previously reported that delphinidin, a com-
pound that possesses pharmacological properties
similar to those of RWPC, inhibits endothelial cell
proliferation; this effect is associated with an increase
of caveolin-1 expression, which might decrease eNOS
activation and therefore exert a negative regulatory
effect on proliferation on either endothelial cells
(15) or transformed cells such as NIH-3T3 cells (31).
RWPCs contained a large number of compounds,
including phenolic acids, flavonoids, anthocyanins,
and tannings. Since Provinols™ is a mixture of
different polyphenolic compounds, it is not certain
which polyphenolic components are responsible for
the pro- or anti-angiogenic process. To further ad-
dress this question, we performed additional experi-
ments with delphinidin. We showed that a high dose
of delphinidin decreased revascularization after 15
days of ligature. Consequently, we demonstrated that
delphinidin is probably the key compound (or one of
them) implicated in the anti-angiogenic effect of
RWPC. Our previous study showed that the antipro-
liferative effect of delphinidin occurs independent of
NO pathway. With regard to Akt/PI3K pathways,
polyphenols from tea have been reported to decrease
the expression of PI3K and Akt phosphorylation in
human prostate cancer cells (32). We showed here
that inhibition of Akt/PI3K also took place in isch-
emic legs after a high dose of RWPC. A high dose of
RWPC inhibited either the expression or the activity
of two major proangiogenic factors, VEGF and
MMPs, in association with reduced P-p38 expression.
These results are in accordance with those reported
in cultured smooth muscle cells in which redox-
sensitive inhibition of the p38 MAPK pathway activa-
tion leads to inhibition of PDGF-induced VEGF ex-
pression (17) and redox-insensitive mechanisms lead
to inhibition of thrombin-induced MMP-2 formation
(18). Our results also strengthened those reported by
Barthomeuf et al. (33) showing that red grape skin
polyphenols inhibit angiogenesis in a Matrigel model
and reverse the chemotactic effect of VEGF on
bovine aortic endothelial cells. This inhibition is
associated with down-regulation of ERK1/2 and p38
phosphorylation. Finally, a high dose of RWPC inhib-
ited NF-B expression in the ischemic legs. Polyphe-
nols from different sources such as tea or hop plant
have been reported to possess anti-angiogenic prop-
erties: they inhibit growth of a vascular tumor in vivo
and repress the NF-B and Akt pathways in endothe-
lial cells (34). To the best of our knowledge, our
study extended the participation of NF-B and Akt
pathways in the anti-angiogenic effect of RWPC.
Altogether, our finding provides a rational explana-
tion for tumor growth inhibition and the anti-athero-
sclerotic effect of RWPC used at a high dose, although
no study had yet clearly demonstrated their anti-angio-
genic effect in ischemic conditions, especially in vivo.
3519RED WINE POLYPHENOLS, DUAL EFFECTS ON ANGIOGENESIS
The anti-angiogenic effect of RWPC is being investi-
gated using in vitro models (i.e., endothelial cell) or
tumor cell growth in culture (35) or in vivo studies
predominantly performed in Matrigel and CAM mod-
els (33).
Finally, and most interesting, we show for the first
time that a low dose of RWPC promoted angiogenesis
in ischemic conditions in association with increased
blood flow and arteriolar density. A low dose of
RWPC did not affect capillary density, suggesting that
RWPC affect arteriogenesis rather than angiogenesis
(Fig. 2B) (36). In our study, the capillary density was
evaluated in the bottom of the thigh after 15 days of
ligature, which could explain that the capillary den-
sity was not affected by the treatment. Furthermore,
Deindl et al. (37) have demonstrated that VEGF was
not implicated in arteriogenesis. In our study, the low
dose of RWPC increased VEGF expression. In addi-
tion, a neutralizing VEGF antibody inhibited neovas-
cularization induced by a low dose of RWPC (Fig. 8).
Consequently, our results demonstrated the involve-
ment of the VEGF pathway in the proangiogenic
effect of polyphenols. Indeed, these results underline
that the low dose of RWPC possesses proangiogenic
rather than proarteriogenic properties. The mecha-
nism involved activation of NO, Akt/PI3K, p38
MAPK, and VEGF expression without alteration in
either MMP activity or NF-B expression. Thus, a low
circulating level of RWPC able to improve postisch-
emic neovascularization was present in vivo.The
nature of these compounds has not yet been assessed.
Anthocyanins such as delphindin and oligomeric-
condensed tanning exhibit a pharmacological profile
comparable to original RWPC in terms of endothelial
NO release (38); alternatively, they could activate
eNOS, Akt/PI3K, and VEGF. Consequently, we have
performed additional experiments in order to test
the effect of delphinidin in our study (Fig. 9). We
found that angiogenesis was not affected by a low
dose of delphinidin whereas a high dose decreased
the revascularization. Consequently, we demon-
strated that delphinidin is probably one of the com-
pounds implicated in the anti-angiogenic effect of
RWPC. These data also favor in the hypothesis that
the differential effect obtained between a low and a
high dose of RWPC might be related to its composi-
tion.
RWPC stimulate endothelial NO production through
an increase in intracellular calcium levels and through
activation of the PI3-kinase/Akt pathway in endothelial
cells (9-11). RWPC may also regulate NO activity at the
level of eNOS protein expression in endothelial cells
(12), blood vessels, and cardiac tissues (13). In addition
to the NO pathway, we showed the capacity of a low
dose of RWPC to increase VEGF expression. Alto-
gether, a low dose of RWPC and, consecutively, mod-
erate consumption of red wine could have a beneficial
effect in ischemic diseases requiring angiogenesis
and/or an increase in blood flow.
CONCLUSIONS
The present study highlights a dose-dependent effect of
polyphenols in an in vivo model of angiogenesis trig-
gered by ischemia. The results give credence to the
notion that RWPC exert a unique dual effect and offer
important therapeutic perspectives for the treatment
and prevention of ischemic diseases (low dose) or
cancer growth (high dose). Indeed, RWPC is orally
active, with no potentially serious adverse effects and
high degree tolerability, and therefore a good safety
profile in addition to the low cost of their use.
C.B.-M. and A.B. are fellows of the Pays de la Loire Region,
France. We thank the local Animal Care Unit of the Univer-
sity of Angers and Je´roˆme Roux, Pierre Legras, and Domin-
ique Gilbert for their kind help in treating the rats. We thank
Mlle. Nowicki Marion for technical assistance.
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Received for publication November 30, 2006.
Accepted for publication May 24, 2007.
3521RED WINE POLYPHENOLS, DUAL EFFECTS ON ANGIOGENESIS
