Advance Access Publication 27 October 2007 eCAM 2010;7(1)47–56
Vaccinium myrtillus (Bilberry) Extracts Reduce Angiogenesis
In Vitro and In Vivo
Nozomu Matsunaga1, Yuichi Chikaraishi1, Masamitsu Shimazawa1, Shigeru Yokota2
and Hideaki Hara1
1Department of Biofunctional Evaluation, Molecular Pharmacology, Gifu Pharmaceutical University, 5-6-1
Mitahora-higashi, Gifu 502-8585 and2Wakasa Seikatsu Co. Ltd, 22 Naginataboko-cho, Shijo-Karasuma,
Shimogyo-ku, Kyoto 600-8008, Japan
Vaccinium myrtillus (Bilberry) extracts (VME) were tested for effects on angiogenesis in vitro and in
vivo. VME (0.3–30mgml?1) and GM6001 (0.1–100mM; a matrix metalloproteinase inhibitor)
concentration-dependently inhibited both tube formation and migration of human umbilical vein
endothelial cells (HUVECs) induced by vascular endothelial growth factor-A (VEGF-A).
In addition, VME inhibited VEGF-A-induced proliferation of HUVECs. VME inhibited
VEGF-A-induced phosphorylations of extracellular signal-regulated kinase 1/2 (ERK 1/2) and
serine/threonine protein kinase family protein kinase B (Akt), but not that of phospholipase Cg
(PLCg). In an in vivo assay, intravitreal administration of VME inhibited the formation of
neovascular tufts during oxygen-induced retinopathy in mice. Thus, VME inhibited angiogenesis
both in vitro and in vivo, presumably by inhibiting the phosphorylations of ERK 1/2 and Akt.
These findings indicate that VME may be effective against retinal diseases involving angiogenesis,
providing it can reach the retina after its administration. Further investigations will be needed to
clarify the major angiogenesis-modulating constituent(s) of VME.
Keywords: angiogenesis–VEGF–Bilberry (Vaccinium myrtillus) extraction–GM6001–MMP–
Angiogenesis is the process by which blood vessels are
formed from pre-existing ones. In adults, physiological
angiogenesis is observed only at restricted sites, such as
the endometrium and ovarian follicle, and it is normally
many ocular diseases, such as diabetic retinopathy (1),
age-related macular degeneration (2) and neovascular
glaucoma(3). Previous studies
angiogenesis is explicitly increased by several growth
factors, such as VEGF (4), basic fibroblast growth factor
(5) and platelet-derived growth factor (6).
Galardy et al. (7) reported that a carcinoma extract
implanted in the rat cornea can be used to stimulate
angiogenesis from the vessels of the limbus, and also that
continuous administration of GM6001, a broad-spectrum
matrix metalloproteinase (MMP) inhibitor, reduced both
the vessel number and vessel area. More recently, Koike
et al. (8) found that GM6001 decreases tubulogenesis
in microvascular endothelial cells from young humans.
These findings suggest that MMP plays a pivotal role in
angiogenesis, and that MMP inhibitors may be effective
Ericaceous family, can be found in the mountains and
forests ofEuropeand North
a memberof the
For reprints and all correspondence: Professor H. Hara, PhD,
Department of Biofunctional Evaluation, Molecular Pharmacology,
Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585.
Japan. Tel: +81-58-237-8596; Fax: +81-58-237-8596;
? 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/
licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is
anthocyanins (9,10) have been shown to possess potent
antioxidant properties (9), stabilize collagen fibers and
promote collagen biosynthesis (11) and inhibit platelet
aggregation (12). Animal studies have demonstrated
VME to be of benefit in improving vascular tone,
blood flow and vasoprotection (13,14). When adminis-
tered to healthy subjects or to patients with visual
disorders, VME (either alone or in combination with
b-carotene and vitamin E) induces a significant improve-
ment in night vision, a quicker adaptation to darkness
and a more rapid restoration of visual acuity following
exposure to a flash of bright light (11). Hence, bilberries
(or VME) have been utilized as a popular edible aid
or supplement for asthenopia and improved visual
function. Furthermore, an extract of V. myrtillus fruits
(a low concentration of anthocyanosides in a highly
purified extract) has been reported to induce significant
improvements in ophthalmoscopic and angiographic
images in diabetic or hypertensive patients (15), but it
has remained unclear whether it inhibits angiogenesis.
Roy et al. (16) noted that in the human keratinocytes
cell-line HaCaT, VEGF expression is decreased by a
variety of berry seeds, such as bilberry, raspberry,
strawberry, blueberry and optiberry (a blend of wild
blueberry, strawberry, cranberry and raspberry seeds,
and elderberry and wild bilberry samples). They also
observed that optiberry inhibits the tube formation
among human microvascular endothelial cells induced
by basement proteins from mouse tumors. These findings
suggest that certain berry seeds have inhibitory actions
against angiogenesis, although, the precise mechanism
remains unclear. We therefore examined the in vitro
effects of VME on the angiogenesis (tube formation, and
cell proliferation and migration) and phosphorylation
of extracellular signal-regulated kinase 1/2 (ERK 1/2),
phospholipase Cg (PLCg) and serine/threonine protein
kinase family protein kinase B (Akt) that are induced by
vascular endothelial growth factor-A (VEGF-A). We also
evaluated the in vivo effects of VME on oxygen-induced
retinopathy in mice.
were purchased from Sigma (St. Louis, MO, USA)
and Kurabo (Osaka, Japan), respectively. The oxygen-
scavenger N-acetyl-L-cysteine (NAC) and Trolox, a solu-
ble vitamin E derivative, were purchased from Wako
(Osaka, Japan) and Sigma, respectively. Antibodies
against phosphorylated ERK 1/2 (Thr 202/Tyr 204),
total ERK 1/2, phosphorylated PLCg (Tyr 783) and total
PLCg were purchased from Cell Signaling Technology
(Beverly, MA, USA). An antibody against b-actin was
purchased from Sigma. VME were purchased from
Fushimi Chemical Co., Ltd (Kyoto, Japan). It was
extracted in accordance to a method as previously
described by Nakajima et al. (9). Briefly, VME were
extracted from commercially available paste frozen fruits
of bilberry using ethanol, filtrated and concentrated.
Then ethanol extracts of bilberry were applied to column
chromatography, removed ethanol and freeze-dried to
powder. Fifteen kinds of anthocyanin components of
VME were ascertained by use of high-pressure liquid
chromatography. VME were containing 25% anthocyanin
(conversion of anthocyanin into delphinidin), and 15 kinds
pyranoside, Delphinidin 3-O-glucopyranoside, Cyanidin
3-O-galactopyranoside, Delphinidin 3-O-alabinopyrano-
side, Cyanidin 3-O-glucopyranoside, Petunidin 3-O-galac-
topyranoside, Cyanidin 3-O-alabinopyranoside, Petunidin
3-O-glucopyranoside, Paeonidin 3-O-galactopyranoside,
Petunidin 3-O-alabinopyranoside, Paeonidin 3-O-gluco-
pyranoside, Malvidin 3-O-glucopyranoside, Paeonidin
3-O-alabinopyranoside, Malvidin 3-O-galactopyranoside
and Malvidin 3-O-alabinopyranoside, respectively.
(Hamamatsu, Japan). All mice were handled according
to the ARVO statement for the Use of Animals in
Ophthalmic and Vision Research, and the experiments
were approved and monitored by the Institutional
AnimalCare and Use
Kurabo) were cultured in a growth medium (HuMedia-
EG2; Kurabo) at 37?C in a humidified atmosphere of
5% CO2in air. The HuMedia-EG2 medium consists of a
base medium (HuMedia-EB2, Kurabo) supplemented with
2% fetal bovine serum (FBS), 10ngml?1recombinant
human epidermal growth factor, 1mgml?1hydrocortisone,
5ng ml?1recombinant human basic fibroblast growth
factor -B and 10mgml?1heparin.
umbilicalvein endothelialcells (HUVECs,
Tube Formation Assay
An angiogenesis assay kit (Kurabo) was used according
to the manufacturer’s instructions. Briefly, HUVECs
presence or absence of various concentrations of test
drugs plus VEGF-A (10ngml?1). After 11 days, cells
were fixed in 70% ethanol. The cells were incubated with
diluted primary antibody (mouse anti-human CD31,
1:4000) for 1h at 37?C, and with the secondary antibody
(goat anti-mouse IgG alkaline phosphatase-conjugated
were cultivated inthe
48V. myrtillus extracts reduce angiogenesis
antibody, 1:500) for 1h at 37?C, and visualization was
achieved using 5-bromo-4-chloro-3-indolyl phosphate/
nitro blue tetrazolium. Images were obtained from five
different fields (5.5mm2per field) for each well, and tube
area, length, joints and paths were quantified using
Angiogenesis Image Analyzer Ver.2 (Kurabo).
Cell Proliferation Assay
HUVECs were seeded into 96-well plates at a density
2000 cells per well at 37?C for 12h, and preincubated
in HuMedia-EB2 containing 2% FBS at 37?C for 6h.
The HUVECs were incubated for 48h in fresh medium
containing VEGF-A (10ngml?1) with or without various
concentrations of test drugs, and then incubated for a
further 48h in the same (fresh) medium. After incuba-
tion, the viable cell numbers were measured by means
of a WST-8 assay. Briefly, 10ml of CCK-8 (Dojindo,
Kumamoto, Japan) was added to each well, incubated at
37?C for 3h and the absorbance measured at 492nm
(reference wave, 660nm).
Cell Migration Assay
Cell migration was evaluated using a modified Boyden
chamber assay (17). The microporous membrane (8mm)
of 24-well cell-culture inserts (BD Bioscences, Bedford,
MA, USA) was coated with human fibronectin (BD
Bioscences). HUVECs were collected by centrifugation,
resuspended in HuMedia-EB2 containing 0.1% bovine
serum albumin (BSA) and seeded into the chamber
HuMedia-EB2 containing 0.1% BSA and VEGF-A
(10ngml?1) with or without test drugs, and the chamber
was incubated at 37?C for 4h in 5% CO2. Any migrated
cells on the upper surface of the membrane were removed
by scrubbing with a cotton swab. Migrated cells on the
lower surface of the membrane were fixed in Diff-Quik
Fixative(Sismex, Kobe, Japan)
hematoxylin. The migrated cells were then counted in
five fields (for each membrane) under a microscope at
?200 magnification and the average number per field was
cells per well). Each well was filled with
Subconfluent HUVECs were incubated in HuMedia-EB2
containing 2% FBS for 6h at 37?C in a 5% CO2
Dulbecco’s modified Eagle medium containing 25mM
(Invitrogen, Grand Island, NY, USA) and either 2%
FBS or 0.5% FBS (for Akt detection), and incubation
allowed to proceed for a further 1 or 18h, respectively, at
37?C. Next, the medium was changed to fresh medium
(constituents as above) containing VEGF-A (10ngml?1)
concomitantly with or without VME (30mgml?1), and
incubation continued for 5 or 10min (we performed pilot
study for time course of changes in phosphorylated –
ERK 1/2 and Akt after VEGF treatment, and they were
the highest at 5 and 10min after that, respectively). The
HUVECs were washed two times with 10mM NaF in
PBS, lyzed in RIPA buffer (Sigma) supplemented with
protease inhibitor cocktail (Sigma), phosphatase inhibitor
cocktail 1 (Sigma) and phosphatase inhibitor cocktail
2 (Sigma), and stocked at ?80?C. Equal amounts of each
sample were electrophoresed on 7.5% SDS–PAGE gel,
then transferred to polyvinylidene difluoride membranes.
After blocking with Blocking One-P (Nacarai tesque,
Kyoto, Japan) for 30min, the membranes were incubated
with one of the following, as the primary antibody:
phosphorylated Akt, anti-total Akt or anti b-actin
antibody. After this incubation, the membrane was
incubated with secondary antibody: HRP conjugated
goat anti-rabbit or -mouse IgG (Pierce Biotechnology,
Rockford). The immunoreactive bands were visualized
using Super Signal?West Femto Maximum Sensitivity
Substrate (Pierce Biotechnology) and measured using
GelPro (Media Cybernetics, Silver Spring, MD).
Oxygen-induced Retinopathy in Mice
Oxygen-induced retinopathy was induced in newborn mice
as previously described by Smith et al. (18) Briefly, on
post-natal day 7 (P7) mice were placed along with their
dam into a custom-built chamber in which the partial
pressure of oxygen was maintained at 75%. Mice were
maintained in 75% oxygen for up to 5 days (P12), after
which they were transferred back to their cage in room air.
VME (300ng per eye) or saline was intravitreously injected
on P12. Mice were anesthetized by intraperitoneal admin-
istration of sodium pentobarbital salt (Dainippon sumi-
tomo pharma, Osaka, Japan) at 50mgkg?1. Through a
median sternotomy, the left ventricle of the heart was
identified and perfused with FITC dextran (20mg per
animal). Then, the eyes were enucleated and placed in
4% paraformaldehyde. Under a dissecting microscope,
cutting and covered with a coverslip after a few drops
Laboratories, Burlingame, CA) had been placed on the
slide. Images of flat-mounted retinas were acquired via a
fluorescence microscope (BX50; OLYMPUS, Tokyo,
Japan) using a high-resolution charged-coupled device
camera (DP30BW; OLYMPUS, Tokyo, Japan). The areas
of neovascular tufts in the retina were measured using
imaging software (Metamorph; Universal Imaging Corp.,
eCAM 2010;7(1) 49
Measurement of 2,2-diphenyl-1-picrylhydrazyl
Radical-scavenging activity was measured by means of a
2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (19). VME,
NAC and Trolox were dissolved and diluted in ethanol at
various concentrations and then 0.025mgml?1DPPH in
ethanol was added, and the whole left to stand for 30min
at room temperature. This was followed by measurement
of the absorbance of the resulting solution at 517nm
using a spectrophotometer.
Measurement of Lactate Dehydrogenase Activity
Lactate dehydrogenase (LDH) activity in the culture
medium containing VEGF with or without VME at
30mgml?1(the highest dose in this study) was measured
using an LDH cytoxicity Detection kit (Takara Bio,
Data are presented as means?SEM. Statistical com-
parisons were made using a one-way ANOVA followed
by a Student’s t-test, paired t-test or Dunnet’s multiple-
comparison test. A value of P<0.05 was considered to
indicate statistical significance.
VME Inhibited VEGF-A-induced Tube Formation in
HUVEC Co-cultured with Fibroblast
Representative images of tube formation induced by
VEGF-A with or without VME are shown in Fig. 1A.
VME (0.3–30mgml?1) concentration-dependently inhib-
ited tube formation and quantitative analysis showed that
VME at 1–30mgml?1significantly inhibited tube area,
length, joints and paths (Fig. 1B–E). At 3mgml?1or
0.33 10 301
% of control
% of control
% of control
% of control
Figure 1. VME inhibited tube formation induced by VEGF-A. Representative photographs of tube formation (A). Scale bar=100mm. HUVECs
were co-cultured with human fibroblasts, as described in Methods section, and incubated for 11 days with or without the indicated concentrations of
VME, with the concomitant addition of VEGF-A (10ngml?1). Tube formation was observed in five randomly chosen fields, and tube area (B),
length (C), joints (D) and paths (E) were measured using an Angiogenesis Image Analyzer. Data are shown as mean?SEM (n=3–6). C: Control.
*, P<0.05; **, P<0.01 versus VEGF-A (Dunnett’s multiple-comparison test). ***, P<0.01 versus Control (Student’s t-test).
50V. myrtillus extracts reduce angiogenesis
more, VME reduced all four parameters to the non-
treated control level (Fig. 1B–E).
GM6001 Inhibited VEGF-A-induced Tube Formation in
HUVEC Co-cultured with Fibroblast
GM6001 (10–100mM) significantly inhibited
A-induced tube formation in a concentration-dependent
manner (Fig. 2A–D). The highest concentration of
GM6001 used (100mM) reduced tube formation to the
non-treated control level (Fig. 2A–D).
VME Inhibited VEGF-A-induced HUVEC Proliferation
Cell proliferation in HUVECs was increased to ?200%
of control by VEGF-A (10ngml?1) treatment (Fig. 3).
VME (3–30mgml?1) inhibited this proliferation in a
concentration-dependent manner, its effect being signifi-
cant at 3mgml?1or more (Fig. 3A). On the other hand,
VME alone (without VEGF-A) had little or no effect
on basal proliferation (Fig. 3A).
VME and GM6001 Inhibited VEGF-A-induced
Cell migration in HUVECs was increased to 190% of
control by VEGF-A (10ngml?1) treatment (Fig. 4).
VME (3–30mgml?1) inhibited
concentration-dependent manner, its effect being signifi-
cant at both 10 and 30mgml?1(Fig. 4B). On the other
hand, VME (30mgml?1) alone had no effect on HUVEC
migration (versus control) (Fig. 4B). GM6001 (3–30mM)
significantly inhibited the HUVEC migration induced
by VEGF-A (10ngml?1).
VME Inhibited VEGF-A-induced Phosphorylation of
ERK 1/2, but not that of PLCc
We analyzed the effects of VME on the signaling
pathways induced by VEGF-A. Activation of ERK 1/2
and PLCg has been reported to be involved in VEGF-
induced proliferation (20). Treatment with VEGF-A
(10ngml?1) for 5min increased the phosphorylation of
% of control
0.11 10 100
% of conxtrol
% of control
% of control
0.11 10 100
Figure 2. GM6001 inhibited tube formation induced by VEGF-A. HUVECs were co-cultured with human fibroblasts, as described in Methods
section, and incubated for 11 days with or without the indicated concentrations of GM6001, with the concomitant addition of VEGF-A (10ngml?1).
Tube formation was observed in five randomly chosen fields, and tube area (A), length (B), joints (C) and paths (D) were measured using an
Angiogenesis Image Analyzer. Data are shown as mean?SEM (n=4–8). C: Control. **, P<0.01 versus VEGF-A (Dunnett’s multiple-comparison
test). ***, P<0.01 versus Control (Student’s t-test).
eCAM 2010;7(1) 51
ERK 1/2 (p-ERK 1/2) approximately 2.5-fold and the
5.2-fold (Fig. 5A and B). VME (30mgml?1) significantly
inhibited the VEGF-A-induced increase in p-ERK 1/2
(Fig. 5A), but not that in p-PLCg (Fig. 5B). VME
(30mgml?1) had no effects on either p-ERK 1/2 or
p-PLCg (Fig. 5A and B).
VME Inhibited VEGF-A-induced Phosphorylation of Akt
Activation of Akt is known to be an important step
in the VEGF-induced migration of HUVECs (21,22).
increased the phosphorylation of Akt (p-Akt) approxi-
mately 1.5-foldand VME
inhibited this increase (Fig. 5C).
(10ngml?1) for 10min
3 10 30
Migration (% of control)
Figure 4. VME and GM6001 inhibited migration induced by VEGF-A. Representative photographs showing effects of VME and GM6001 on
HUVEC migration (A). Scale bar=50mm. The migrated cells were counted in five fields for each membrane (see Methods section) (B). Data are
shown as mean?SEM (n=5 or 6). C: Control. **, P<0.01 versus VEGF-A (Dunnett’s multiple-comparison test). ***, P<0.01 versus Control
0.313 10300.313 1030
Proliferation (% of control)
Figure 3. VME inhibited proliferation of HUVECs induced by VEGF-A. HUVECs were cultured in 96-well plates at a density of 2000 cells per well,
then incubated for a total of 96h at 37?C in 5% CO2. HUVECs were supplemented with or without VEGF-A (10ngml?1) plus various
concentrations of test drugs, and measurements were made by WST-8 assay. Data are shown as mean?SEM (n=5–8). C: Control. *, P<0.05;
**, P<0.01 versus VEGF-A (Dunnett’s multiple-comparison test). ***, P<0.01 versus Control (Student’s t-test).
52 V. myrtillus extracts reduce angiogenesis
VME Inhibited Angiogenesis during Oxygen-induced
Retinopathy in Mice
The excessive neovascularizaion observed in flat-mounted
retinal sections obtained from mice after prolonged
exposure to 75% oxygen was estimated by analysis of
the vascular tufts. Representative images of such neo-
vascularization in mice treated with or without VME are
shown in Figs 6A and B. Intravitreal administration of
VME (300ng per eye) significantly inhibited the area
of the neovascular tufts (versus vehicle) (Fig. 6C).
VME and Anti-oxidants Exhibited Radical-scavenging
Activity against DPPH Radical
The radical-scavenging activity of VME was compared
with those of NAC and Trolox using a DPPH assay. As
shown in Table 1, VME, NAC and Trolox concentration-
dependently exhibited radical scavenging ability against
DPPH radical, the IC50values being 9.1mgml?1, 23.1mM
and 24.1mM, respectively.
LDH Activity in HUVEC Culture Medium was not
Significantly Increased by Treatment of VME
We measured LDH activity in culture medium to
examine cytotoxicity ofVME.
(n=6) in VEGF-treated medium, and those activity of
LDH were not significantly different.
In the present study, we found that VME inhibited
angiogenesis both in vitro and in vivo, and our results
suggest that its effect may be due in part to reductions in
cell proliferation and migration through inhibition of
both p-ERK 1/2 and p-Akt.
Angiogenesis is a multi-step process, and VEGF
promotes many of the events necessary for angiogenesis,
such as proliferation and migration of endothelial cells,
remodeling of extracellular matrix and formation of
capillary tubules (19). Extracellular-matrix degradation is
critical during angiogenesis, which requires proteolysis
of endothelial cells as well as synthesis of new matrix
mediated by specific proteases called MMP, which are
smooth muscle cells and reportedly also by myocytes
(23,24). Furthermore, in vitro and in vivo studies have
shown that MMP inhibitors (GM6001 and TIMP1, a
tissue inhibitor of metalloproteinase-1) decrease the
angiogenesis induced by VEGF (7,25,26). In the present
study, GM6001 inhibited VEGF-A-induced tube forma-
tion in HUVECs co-cultured with human fibroblast cells
of matrixcomponents is
Figure 5. VME inhibited phosphorylations of ERK 1/2 and Akt
induced by VEGF-A, but not that of PLCg. Effects of VME
(30mgml?1) on VEGF-A (10ngml?1)-induced ERK 1/2 (A), PLCg
(B) and Akt (C) phosphorylations. Data are shown as mean?SEM
(n=4–7). C: Control.; *, P<0.05; **, P<0.01 versus VEGF-A
(Student’s t-test). ***, P<0.05 versus Control (Student’s t-test).
eCAM 2010;7(1) 53
(Fig. 2). These results suggest that MMP is an important
factor for angiogenesis.
A-induced tube formation (Fig. 1), its effect being
significant at 1–30mgml?1. The antiangiogenic effect of
VME at 3mgml?1was almost equal to that of GM6001at
100mM. It has been reported that a number of berries,
including bilberry, inhibit angiogenesis in vivo (27) as well
as VEGF expression in human keratinocytes in vitro (16).
However, our finding is the first report demonstrating
that VME can inhibit VEGF-A-induced angiogenesis.
When we evaluated the effects of VME on the prolifera-
tion and migration of HUVECs, we found that VME
significantly inhibited VEGF-A-induced HUVEC prolif-
eration, although VME alone had no effect.
GM6001 strongly inhibited VEGF-A-induced HUVEC
migration. This result indicates that the inhibitory effect
of GM6001 on tube formation is mediated by a reduction
in cell migration through a suppression of MMP activity.
According to Lin et al. (28), an antioxidant substance,
HUVECs and its effect is mediated by an inhibition of
the Src (cytoplasmic protein tyrosine kinase) signal
pathway. Furthermore, Ushio-Fukai et al. (29) reported
that VEGF-induced endothelial cell signaling and angio-
genesis is tightly controlled by the reduction/oxidation
environment. Here, VME displayed radical-scavenging
activity (IC50=9.1mgml?1) and significantly inhibited
VEGF-A-induced HUVEC proliferation at concentra-
tions of 3–30mgml?1. Many berry species, including
bilberry, contain a lot of anthocyanins, which possess
antioxidant activities. Therefore, the inhibitory action of
VME on cell migration may be due in part to its
Area of neovascular tufts
Figure 6. VME inhibited neovascular tufts on oxygen-induced retinopathy in mice. Retinal flat mounts were examined by FITC-dextran
angiography. Representative photographs of retina from saline-treated eye (A) and VME-treated eye (B). Scale bar=100mm. Areas of neovascular
tufts in saline- and VME-treated eyes (C). Each column and bar represents mean?SEM (n=9). *, P<0.05 versus Saline (paired t-test).
Table 1. Radical-scavenging activities of VME, NAC and Trolox
Treatments % radical-scavenging
9.1 (7.6–11.0) mgml?1
NAC 3mM 23.1 (21.3–25.1) mM
Trolox 3mM24.1 (22.2–26.3) mM
VME, NAC, and Trolox were incubated with DPPH for 30min, and
the absorbance at 517nm due to DPPH radical was determined. Data
are shown as mean?SEM. (n=5).
54V. myrtillus extracts reduce angiogenesis
Activation of the MAP kinases ERK 1/2 and/or PLCg
is important for the proliferation of HUVECs. We
therefore evaluated the effect of VME on phosphorylated
ERK 1/2 and PLCg. In this study, VME inhibited the
VEGF-A-induced phosphorylation of ERK 1/2, but not
that of PLCg. These results suggest that VME exert
a direct inhibition downstream of PLCg and upstream of
ERK 1/2 in the signaling cascade induced by VEGF-A.
Since activation of Akt is known to be important for the
migration of HUVECs, we also evaluated the effect of
VME on phosphorylated
phosphorylation of Akt induced by VEGF-A. Ali et al.
(30) indicated that PD98059, an ERK 1/2 inhibitor,
inhibits the VEGF-A-induced proliferation of HUVECs,
but not migration, and LY294002, an Akt inhibitor,
inhibits both proliferation and migration. They concluded
that phosphorylation of ERK 1/2 induced proliferation
of HUVECs, and phosphorylation of Akt induced both
proliferation and migration of HUVECs. These findings
suggest that VME reduce the VEGF-A-induced prolif-
eration through inhibiting direct and/or upstream of
ERK 1/2 from downstream of PLCg, and the VEGF-
A-induced proliferation and migration through inhibiting
direct and/or upstream of Akt. However, further studies
are needed to clarify the precise molecular targets
Recently, Sylvie et al. (31) reported that delphinidin,
a kind of anthocyanidin (non-glycosylated form of
anthocyanin), inhibits the VEGF-induced phosphoryla-
tionof ERK1/2, itshalf
achieved at 11.8mM. In the present study, VME inhibited
VEGF-A-induced phosphorylation of ERK 1/2, an
(30mgml?1), as used in this research, contains delphinidin
at ?2mM. Thus, we consider that the inhibitory effect of
VME on the VEGF-A-induced phosphorylation of ERK
1/2 may be mediated by delphinidin and/or other
constituents. Further studies will be needed to identify
the effective constituents of VME.
In our in vivo study, we examined the effect of VME
on angiogenesis using a murine oxygen-induced retino-
(300ng per eye) significantly inhibited the area of
neovascular tufts. Wechose that concentration
VME, because the concentration reached in the vitreous
body was an estimated 30mgml?1. In the present in vitro
analysis, VME at 30mgml?1inhibited the tube formation,
HUVEC proliferation and migration and phosphoryla-
tions of ERK 1/2 and Akt induced by VEGF-A. Taken
together, the above observations suggest that VME
inhibit angiogenesis in vitro and in vivo within the same
range of concentrations. Furthermore, recent research
demonstrated that oxidative stress is associated with
increased production of VEGF under in vitro conditions,
and believed to be an upregulation of VEGF expression
during diabetes (32–34). Collectively, these reports and
Akt. VME inhibitedthe
effect beingachievedat 30mgml?1. VME
administration of VME
our data suggest that the antioxidative effects of VME
may lead to an inhibition of VEGF expression, and that
by this mechanism VME may inhibit VEGF-induced
angiogenesis in the retina.
In conclusion, our findings indicate that VME inhibits
VEGF-induced angiogenesis, and that this effect is
mediated by inhibition of both cell proliferation and
migration. Further experiments will be needed to clarify
the major antiangiogenetic constituents of VME.
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growth factor receptor-2
Received February 6, 2007; accepted September 12, 2007
56 V. myrtillus extracts reduce angiogenesis