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Int. J. Biol. Sci. 2010, 6
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443
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2010; 6(5):443-453
© Ivyspring International Publisher. All rights reserved
Research Paper
Catalpol Increases Brain Angiogenesis and Up-Regulates VEGF and EPO in
the Rat after Permanent Middle Cerebral Artery Occlusion
Hui-Feng Zhu
1
, Dong Wan
2
, Yong Luo
3
, Jia-Li Zhou
4
, Li Chen
5
, Xiao-Yu Xu
1
1. School of Pharmaceutical Sciences & School of Chinese Medicine, Southwest University, Chongqing 400715, China;
2. Department of Emergency Medicine, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,
China;
3. Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China;
4. Chongqing Medical And Pharmaceutical College;
5. Southwest University 2nd Hospital, Chongqing 400715, China
Corresponding author: Professor Xiao-Yu Xu, Laboratory of Molecular Pharmacology, School of Pharmaceutical Science
& School of Chinese Medicine, Southwest University, Chongqing 400715, China. Tel: +8 6 -23-6825-0761; Fax:
+86-23-6825-1225; E-mail: zhfbsci@yahoo.cn; xxy0618 @ s i n a .com
Received: 2010.05.24; Accepted: 2010.08.01; Publish ed: 2010.08.20
Abstract
To investigate the role and mechanism of catalpol in brain angiogenesis in a rat model of
stroke , th e ef fect of catal pol (5 mg/kg; i.p) or veh icle administered 24 hours aft er p erma nent
middle cerebral artery occlusion (pMCAO) on behavior, angiogenesis, ultra-structural inte-
grity of brain capillary endothelial cells, and expression of EPO and VEGF were assessed.
Repeated treatments with Catalpol reduced neurological deficits and significantly improved
angiogenesis, while significantly increasing brain levels of EPO and VEGF without worsening
BBB edema. These results suggested that catalpol might contribute to infarcted-brain angi-
ogenesis and ameliorate the edema of brain capillary endothelial cells (BCECs) by upregulating
VEGF and EPO coordinately.
Key words: Catalpol, VEGF, EPO, Permanent occlusion of middle cerebral artery, Angiogenesis
1. Introduction
Stroke has been emerging as one of the most
c o m m o n c a u s e s o f m o r t a l i t y a n d m o r b i d i t y i n m o d e r n
society. A l t h o u g h m u c h p r o g r e s s h a s b e e n m a d e t o-
ward understanding the mechanistic basis of stroke,
the effectiveness of dru gs availa ble for stroke patients
is limited. Tissue plasminogen a ct i v a t o r ( T P A) , w h i c h
di ss olves bl ood c lots in the b rain, is pre se ntly th e on ly
approved treatment for stroke; however, it is effective
only in the first 3 h after the stroke and may lead to
cerebral hemorrhage [1]. Many drugs focus on the
isc he mic pe numb ra and c asc ad e of d ama ge , in cl uding
anti-N-meth y l -D-aspartate receptor (Aptiganel and
gavestinel), potassium channel agonists (MaxiPost),
and GABA modulators (Zendra). However, the uses
of these drugs for stroke have been abandoned be-
cause they are not effective and even harmful to
stroke patients, despite their apparent effectiveness in
animal models of brain ischemia [2]. Therefore, new
drugs a r e in demand an d need to be developed t o
treat stroke.
The neurovascular unit concept emphasizes not
only the neuron but also the brain vascular structure
[3-4]. Previous research on stroke has largely focused
on neuroprotection, but neglected the ischemic vas-
cular structure and the possible benefits of its func-
tional reconstruction [1,4,5]. Brain vascular structures
are coupled with brain neurons in structure and
function [6]. Angiogenesis is associated with neuro-
genesis [7]. Mounting evidence has shown that vas-
cular-remodeling occurs after st ro k e [ 8 -9]. It has been
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shown that higher blood vessel counts correlate with
longer survival in stroke patients [ 10 ] . T h e
three-dimensional images of angiogenesis in the tis-
sue surrounding focal brain infarcts are demonstrated
by scanning electron microscopy with corrosion cast-
ing [11]. These studies suggest that targeting brain
vascular-remodeling for drug discovery is necessary
for stroke and even for ischemia disease.
Rehmannia Root is the main natural herbal
medicine that plays an important role in treat in g
stroke. Recently, catalpol, a main active component of
Rehmannia Root, was determined to pose extensive
ischemic neural protection, such as preventing the
loss of hippocampal CA1 neurons and reducing
working errors [12], modulating the expressions of
Bcl-2 and Bax [13], attenuating apoptosis in the
ischemic brain [13], and increasing hippocampal
neuroplasticity by up-regulating PKC and BDNF in
aged rats [14]. Catalpol improves Y-maze perfor-
mance and the survival of neurons in the CA1 sub-
field after transient global ischemia in gerbils [15-16] .
Our previous studies have demonstrated that catalpol
at doses of 10 and 5 mg/kg can improve neurobeha-
vioral outcome following permanent focal cerebral
ischemia in Sprague Dawley rats, and upregulate the
expression of growth-associated protein 43 (GAP-43)
[17]. These findings suggest that catalpol contributes
to neuroplasticity after stoke. However, whether cat-
alpol can modulate brain angiogenesis after focal
ischemia is unclear.
Erythropoietin (EPO) and Vascular en dothel ial
g r o w t h f a c t o r ( V E G F ) h a v e p l e i o t r o p i c e f f e c t s o n b r a i n
function, including neuroprotection, and promotion
of angiogenesis and neurogenesis [18]. Notably, EPO
enhances angiogenesis, without aggravating brain
edema, e ven u se d wi th VEG F [19].
In this study, we have investigated the effect of
catalpol on angiogenesis following permanent middle
cerebral artery occlusion (pMCAO) in rats. In order to
explore the cellular and molecular mechanism by
which catalpol may regulate the vascular plasticity of
the brain, we examined the expression of VEGF and
EPO by immunohistochemistry and western blotting.
2. Materials and Methods
2.1 Animals and diets
Healthy male Sprague-Dawley (SD) rats (220∼
280 g) were obtained from the Experimental Animal
Center, Chongqing University of Medicine, China.
Animals were housed under conditions of natural
illumination with food and water available ad libi-
tum. These experiments were performed in accor-
dance with China’s guidelines for care and use of la-
boratory animals. An i m a l s w e r e d i v i d e d i n t o 3 g r o u p s
r a n d o m l y ( 1 ) t h e s h a m o p e r a t e d g r o u p ( n = 2 4 ) ; ( 2 ) t h e
vehicle group (n = 24); (3) the catalpol-treated group
(n = 24).
2.2 The pMCAO model
Strokes were induced by electrocoagulation of
the right middle cerebral artery as d es c ri b ed p r e-
viously with minor modifications [20]. B rie fly, rats
were anesthetized and placed in a stereotaxic instru-
ment (Shanghai Jiangwan) in the prone position. The
scalp was opened and brain was exposed, held up
lightly with a glass retractor, inferior cerebral vein
and olfactory bundle were seen perspicuously and the
right middle cerebral artery was along the brain sur-
face verticality striding over inferior cerebral vein and
olfactory bundle. The middle cerebral artery ventral
to the olfactory tract was electrocoagulated (power
35W), resulting in infarction of the right dorsolateral
cerebral cortex. Rats were prescreened to select those
in line with the criteria described as Bederson [21].
2.3 Drug administration
Catalpol was dissolved in physiological saline,
which was purchased from National Institute for the
Control of Pharmaceutical and Biological Products
(China) and its purity was more than 98%. Catalpol
(5mg/kg, ip) were administered 24h after stroke and
then daily for 7 days. Likewise, the sham-operation
group and the vehicle group received equal volumes
of physiological saline by ip injection. The dose of
catalpol was based on our previous study [17] and
Li’s study [15-16].
2.4 Bederson’s Score
After operation, the neurological function of all
animals was evaluated daily with a 4-point scale as
previously described [21]: (0) no apparent deficit, (1)
contr ala teral f ore limb flexion , (2) lowe red r esista nce
to lateral push without circling, and (3) circling to
ipsilateral stroke if spontaneous activity.
2.5 B e a m -Walking test
Beam walking test [22-23] was used to evaluate
sensorimotor reflexes, motor strength and coordina-
tion. The testing apparatus was a 2.5 cm in diameter
and 80 cm in length wooden beam elevated 100 cm
a b o v e t h e f l o o r w i t h w o o d e n supports as described by
Stanley et al, and a 5cm thickness foam pillow placed
under the beam avoid getting wound in a fall.
Rats were allowed to walk to a platform located
at the end of the beam and their behaviors were rec-
orded based on the following six cri t e ri a : (0) the rat
traverses the beam without falling down; (1) the rat
traverses the beam but footslips less than 50%; (2) the
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rat crosses the beam but footslips more than 50%; (3)
t h e r a t c r o s s e s t h e b e a m w i t h o u t t h e a i d o f t h e a f f e c t e d
hindlimb; (4) the rat can’t traverses the beam but can
sit on the horizontal surface of the beam; (5) the rat
will fall down when placed on the beam. Each trial
consisted of five repetitions of this assay.
2.6 Examination of the healing Ischemic brain
cortex and the surface vessels
Stroke model rats in each group were sacrificed
15 days after the operation. After removing the sur-
face meninges, the surface vessels, distribution pat-
terns, and the healing state of ischemic cortex were
examined using a microscope attached to a digital
camera as described previously [24].
2.7 T is sue p re pa ra tio n
Fifteen days after operation/stroke induction,
the rats were deeply anesthetized with an overdose of
Chloral Hydrate (35 mg/mL, i.p), and transcardially
perfused with 0.9 % NaCl solution to rinse out the
blood, followed by 250 mL of 4% formalin (4°C) to fix
the brain tissue. After extraction from the skull, the
b ra i ns we re p os t -fixed in 4% formalin solution and
subsequently cut into 30 µm coronal sections on a
cryostat (Leica). For EPO analyses, imbedded brain
tissue was used. Ipsilateral ischemic cortex (0.1 g per
brain) in each group was also weighed for western
blotting a n aly ses .
2.8 Immunohistochemistry
Animal br a in tissues were transcardially per-
fuse d and fixed with 4% paraformaldehyde, cryopre-
served in 30% sucrose, and cut into three series of
consecutive sections (30 μm) at a Cryostat. Each set of
tissue sections was immunostained for PCNA, V W F
or VEGF. For EPO analyses, imbedded brain tissue
was used to cut as 5μm se ct ions. For immunohisto-
chemistry, tissue sections were incubated with rabbit
polyclonal antibody against EPO (1:200, santa cruz,
USA), VEGF(1:200, Wuhan Booster Biotech. Co.,
China). For double-fluo rescen ce labeli ng, cr oss se c-
tions were incubated with the vessel m a r k e r a n t i b o d y ,
rabbit polyclonal against VWF (1:200, Zhongshan
Biotech. Co., China), together with the mouse mo-
noclonal antibody against the proliferation marker
PCNA (1:200, Wuhan Booster Biotech. Co., China).
Immunohistochemistry for EPO was done with b i o-
tinylated goat anti-rabbit IgG (1:500; Vector Labora-
t or i es ) a nd p e ro xi da s e-conjugated avidin-biot in co m-
plex (ABC kit; Wuhan Booster Biotech. Co., China),
bound antibodies were visualized by addition of di-
aminobenzidine. V E G F s ec t i o ns then incubated with
Cy3-labeled goat anti-rabbit IgG (Wuhan Boster Bio-
tech. Co., China). PC NA a nd VWF then incubated
with Cy3-labeled goat anti-rabbit IgG (Wuhan Boster
Biotech. Co., China) a n d F I T C -labe led goat an -
ti-m o u s e I g G . After through washing, immunostained
ce lls w ere observed under a Nikon microscope and
were documented with a Nikon digital camera. Im-
munofluorescence staining for PCNA (green) and
VWF (red) or VEGF (red) was visualized and docu-
m e n t e d w i t h a c o n f o c a l m i c r o s c o p e (Le i c a) . According
to Acker [25] (Acker et al., 2001), the number of
double-stained vessels and the intensity of staining
were analyzed with Image Pro Plus Version 6.0.
software for cerebral microvessels at the boundary
zone of ischemia. Five fields of each slice were ran -
domly selected for blinded scoring and analyses. E a c h
experiment was performed three times.
2.9 Electron microscopy observation of brain
vascular endothelial cells
Rats in each group were anesthetized with 3.5%
chloral hydrate (35 mg/mL, i.p) and perfused for 2-3
min through the ascending aorta with 0.9% normal
saline, followed by ice-cold fixative (4% paraformal-
dehyde in 0.1 M PBS for 30 min). The brains were
removed for immunocytochemical electron micro-
scopic studies and fixed in 4% paraformaldehyde in
0 . 1 M P B S p H 7 . 4 f o r 3 ～4 h a t 4 ° C . T h e b r a i n w a s t h e n
rinsed in PBS for 30 min, treated with 1% OsO
4
for 30
min, dehydrated in sequential ethanol gradients, and
embedded in Epon 618. Ultrathin sections were
processed according to the postembedding proce-
dure. Briefly, ultramicrocut was made by Leica ul-
tramicrotome to collect 60nm-thick sections. The sec -
tions were mounted on formvar-c o at e d c o pp e r g r id s ,
incubated in uranyl acetate- acetate lead double elec -
t r o n s t a i n , a n d o b s e r v e d f o r b r a i n v a s c u l a r e n d o t h e l i a l
ce lls.
2.10 Western blotting
According to K.N. Na m [26] and Cao Hu ang
[27], brain cortex in peri-ischemic were lysed on ice in
lysi s b uf fer [50 m m T r i s -HC l (pH 8.2), 0.5 M saccha-
rose, 10 mMHEPES (pH 7.9), 1.5 mM MgCl2, 10 mM
KCl, 1 mM EDTA, 10% (v/v) glycerine, 1 mM DTT, 1
mM PMSF, 10μg/mL Aprotinin, and 5μg/mL Leu-
peptin]. After centrifugation at 16,000 ×g for 10 mi-
nutes. Protein content in cleared lysate was deter-
mined by Bradford Assay. Lysate samples containing
40μg of p r o te i n were fractionated by SDS-1 0 % p o-
lyacrylamide gel electrophoresis and then electrob-
lotted onto P CV F membranes. The membranes were
probed with primary antibodies as EPO, VEGF (1:250,
1:300, Satcruze Co., USA) , an d β-a c t i n (1:200, Booster
Biotech. Co., Wuhan, China), then incubated with the
hors e ra d ish peroxidase-conjugated goat anti-mous e
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446
or anti-rabbit IgG (1:2500; Booster Biotech. Co., Wu-
han, China), the PVDF membrane was put into DAB
fluid f o r coloration. Immunoreactivity was di gitall y
scanned b y ScanMaker E6 system a n d quantified us-
ing Q uanti ty one 4. 5.1 ( Bi o-Rad) software. β-ac t i n w a s
used as an internal control for all Western blotting.
2.11 Image and data analysis
After capturing images with a digital camera,
quantification of the results from immunohistoche-
mistry, immunofluorescence, western blotting was
performed with Image Pro Plus Version 6.0. software.
EPO or VEGF-positive cells were counted at five dif-
ferent fields in the inner border of the peri-ischemic
cortex in five sections per rat, the to ta l nu mb er o f EP O
o r V E G F -positive cells per image (cells/cm
2
, o b j e c t i v e
× 20) was calculated by an observer blind to the ex-
perimental treatment. In each section, five pe -
ri-ischemia cortical areas outside labeled neurons
were chosen randomly to obtain an average value for
the subtraction of background by an observer blind to
the experimental treatment.
2.12 Statistical analyses
Data were expressed as mean ± S .E .M. Al l d ata
were analyzed by one-way analysis of variance
(ANOVA) using SPSS 11.0 software. A value of p <
0.05 was co nsider ed sta tistic ally s ignifi cant.
3. Results
3.1 Catalpol improve sensorimotor performance
in stroke rat s
Post-stroke administration of catapol reduced
Bederson's score in rats, indicating improved motor
function re lative to v ehicle c ontrol , catap ol sign ifi-
cantly reduced Bederson's scores in rats at 7 and 15
da ys after s trok e (Fig. 1 A).
The Beam walking test w as us ed to m e as ur e
sensorimotor function. During the course of treat -
ment, beam walking scores were reduced; by day 15
following stroke, score reductions in catapol-treated
versus vehicle-treated animals reached statistical sig-
nificance (P< 0.05) ( Fi g. 1B ).
Figure 1. Effects of intraperitoneal injection with catalpol on sensorimotor performance in post-surgical
rats at days 1, 4, 7 and 15. (A) Post-stroke treatment with catalpol reduced Bederson’s score in stroke rats and (B)
decreased beam working score in stroke rats. T h e d a t a a r e p r e s e n t e d a s m e a n ± SE. * p<0.05 compared with vehicle group,
#
p<0.01 compared with sham operation g ro up.
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3.2 Effect of catalpol on the vascular pattern of
the cerebral cortex surface
The effect of catalpol on the vascular pattern of
the rat cerebral cortex surface was examined follow-
ing pMCAO. In vehicle-treat ed a nimals , li quefa c t i v e
necrosis of the brain was observed 15 days after
p MC AO . B ra in s fr o m vehicle-treated animals exhi-
bited a few deranged microvessels (F ig u re 2A ). B y
contrast, the focus of cerebral ischemia was near
normal in catalpol-treated animals; the brains of thes e
animals exhibited more branched vessels crossing and
gathering radially to the surface of cerebral ischemia.
All these vessels arborized to form a continuous net-
work of small blood vesse l s (F ig ur e 2B ).
Figure 2. T h e vascular pattern in cerebral cortical surface in rats 15 days after pMCAO. (A) In the ve-
hicle-tr e ated group, the pale brain surface had few vessel branch points, infarct areas were characterized by liquefactive
necrosis, cortical surface vessels were scarce and rearranged, several discontinued vascular structures were observed, and
the radial patterns were lost . (B) In the catalpol-treated group, brain surface vessel branch points increased obviously,
vascular structures continued, focus on the infarct area was present, and the vessel radial patterns and brain tissue infarct
area were close to normal. Arrow points to vascular structures around the ischemia area.
3.3 Catalpol enhanced brain angiogenesis in the
peri-infarcted area of the cortex
The effect of catalpol on angiogenesis was then
examined by immunostaining of brain sections for
von Willebrand Factor (vWF), a marker of endothelial
cells, and for proliferating cell nuclear antigen
(PCNA), a marker of cell proliferation. V WF a n d
PCNA co-localization points in the sham o p e ra t i o n
group were scarcly observed (F ig 3A).C o m p a r e d w i t h
the v ehic le-treated g r o u p ( 34 ± 3.25) (Fi g 3B) , t he
number of VWF and PCNA co-localization points in
the catalpol-treated group (Fig 3C an d D ) in c re a se d
significantly (p < 0.05). The number of vWF and
PCNA co-localization points in the catalpol-treated
gr ou p (F ig 3C and D) was 233.67 ± 89.51, nearly 6
times that in the vehicle group (p < 0.01) (Fig 3B an d
D). These results also agreed with results o b ta in e d
from integral optical density (IOD) analyses in the
vWF-P C N A c o -localization area (Fig 3E). These data
demonstrate that catalpol plays an important role in
cerebral ischemia angiogenesis.
3.4 Effects of catalpol on brain capillary endo-
thelial cells (BCECs)
The influence of catapol treatment on BCEC mi-
crostructure following pMCAO were examined by
transmission electron microscopy. Compared to ve-
hi cle co tntrol (F ig 4A), catalpol significantly reduced
BCEC edema (Fig 4B). The number of chondriosomes
in the catalpol-treated group was hi ghe r than that in
the vehicle group and close to normal levels.
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Figure 3. Angiogenesis surrounding ischemic cortical area as demonstrated by immunocytochemistry and
laser scanning confocal microscopy. The effects of Catalpol on angiogenesis were indicated by double-stai ning for
VWF, a marker of endothelial cells and for proliferating cell nuclear antigen (PCNA), a marker of cell proliferation.
Co-labeling of PCNA (green) and VWF (red) demonstrates angiogenesis, i.e., endothelial proliferation in the capillaries, in
the peri-i n f a r c t e d a r e a a t 1 5 d a y s a f t e r p M C A O . C o -localization of P C N A a n d V W F i s y e l l o w . ( A ) S h a m o p e r a t i o n g r o u p , ( B )
Vehicle-treated group, (C) Catalpol-t r e a t e d g r o u p . B a r s = 1 5 0 μm i n A , B , a n d C . T h i s a n a l y s i s d e m o n s t r a t e d that few vessels
were double-s t a i n e d b y V W F a n d P C N A i n s h a m -operated rats (A), but significa n t r e m o d e l i n g o f t h e m i c r o v e s s e l n e t w o r k
o c c u r r e d i n t h e i n f a r c t e d h e m i s p h e r e a n d t h e n u m b e r o f v e s s e l s w i t h s m a l l d i a m e t e r a n d s h o r t s e g m e n t i n c r e a s e d a t 1 5 d a y s
after the stroke. More vessels were double-s t a i n e d f o r V W F a n d P C N A i n c a t a l p o l -treated group (C). Statistical analyses are
s h o w n i n t h e g r a p h o f t h e n u m b e r o f v e s s e l s c o - l a b e l e d f o r P C N A a n d V W F ( D ) . T h e r e s u l t s a b o v e a g r e e d w i t h t h e r e s u l t s
of IOD analyses in the co-localization area (E). (
*
P < 0.01).
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449
Figure 4 Ultrastructural observations of brain capillary endothelial cells (BCECs). In the vehicle-t re at ed gr ou p
( A ) , B C E C e d e m a , c h r o m a t i n r a r e f a c t i o n ( s h o r t a r r o w ) , a n d c h o n d r i o s o m e s w e l l i n g ( l o n g a r r o w ) w e r e o b s e r v e d . ( B ) I n t h e
catalpol group, BCECs, pykno-chromatin (short arrow), and chondriosome number and shape (long arrow) were close to
normal or near normal. Bars = 1μm.
3.5 Catalpol upregulated EPO and VEGF ex-
pression in rat brain following pMCAO
To monitor the influence of catapol on EPO and
VEGF expression, we performed immunohistochem-
ical analyses of brain sections derived from rats fol-
lowing pMCAO. EPO and VEGF- positive cells w er e
detected in the cell membrane and cyt o p l a sm . Fe w
EPO positive cells were observed in brain sections
derived from sham-treated animals ( Fi g 5A). Com-
pared to vehicle-treated animals (Fig 5B), brain sec-
tions derived from catapol-treated animals exhibited
significantly increased EPO expression (Fig 5C). Sta-
tistical analyses revealed 7.2 ± 1.40 positive cell/cm
2
vs 15.3 ± 2 positive cell/cm
2
in vehicle-treated versus
catapol-treated animals (p < 0.05). Similar results were
obtained by western blot analyses (Fi g 5G and I); A s
for V EG F, immunofluorescence analyses demon-
strated that catalpol at the dose of 5mg/ kg signifi-
cantly upregulated VEGF expression compared with
the vehicle group; the number of positive cell/cm
2
vehicle-treated versus catapol-treated animals was 8±
1.6 vs 17 ± 2.5; (p < 0 .01) (F ig . 5E , F, H) . Once again,
similar results were obtained by western blot analyses
(Fig 5G a n d I ) . Statistical analyses were shown in Fig
5I.
4. Discussion
There are three principal findings emerged from
the present study. Firstly, catalpol treatment im -
proved neurofunction after stroke, as evidenced by
enhanced scores in the beam walking test designed to
evaluate sensorimotor reflexes, motor strength and
coordination. Secondl y, o ur resu lts show that catapol
enhances brain angiogenesis following stroke without
wo rs en i ng stroke brain ed e ma . Fina lly, we demon-
strate that t h e ameliorative effects of catalpol on
stroke brain are mediated by enhanced expression of
EPO and VEGF. Our findings thus provide new in -
sights into the likely regulatory mechanisms of cata-
pol.
Ischemic stroke is a serious dis e as e caused by a
thrombus (blood clot), w h i c h c a n r e s u lt i n p e r m an e n t
neurological damage, complications, and even d ea th
[26].Th e standard m e th o d of treatment is t o dissolve
the clot a n d r e s t o r e blood flow in the b l o c k e d vessel.
The drug TP A is approved for this use; however, TPA
is used onl y 3-6 hours after stroke, a n d the more ra-
pidly blood flow is restored to the brain, the fewer
brain cells die [27]. Recent research has suggested that
an alternative approach to restore blood flow is to
promote angiogenesis in regions surrounding the
ischemic brain.
As one of the most potentangiogenic factors,
VEGF is up-regulated by focal cerebral ischemia not
only in animal models but also in human patients
[10-11] as an angiogenic, neurotrophic, and neuro-
protective factor [28-30]. VEGF also plays a vital role
d u r i ng n e u r a l [29,31] and vascular remodeling [32 -33]
after stroke. Our results showed that ischemia in -
duced VEGF expression, which was not enough for
vascular remodeling, but catalpol treatment increased
VEGF expression together with increased microvessel
f or m at i on , he al ed t h e ischemic brain, and improved
neurobehavioral score. These data suggested that cat-
alpol stimulated brain angiogenesis after stroke by
increasing the secretion of endogenous VEGF.
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450
Figure 5 Catalpol upregulated EPO and VEGF expression in pMCAO rat brains. Immunohistochemistry results
showing neurons with EPO (A, B, C, 200×) and VEGF (D, E, F, 200×) in the peri-infarcted area of a pMCAO rat, a
sham-operated rat (A & D), a vehicle-treated rat (B & E), and a catalpol-treated rat (C & F). EPO and VEGF expression
detected by western blot showed in (G). The internal control was β-actin. Vs vehicle group
*
p < 0.05 . The experiments
repeated three times and 6 rats used in eac h group. Statistical bars shown as H and I respectively.
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Moreover, VEGF, known as vascular permeabil-
ity factor, is associated with angiogenesis, vascular
permeability and neuroprotection. VEGF enhances
angiogenesis, but worsens rather than improves ce -
rebral haemodynamics after stroke [34-35]. Lo w-dose
VEGF aggravates hemorrhagic transformation [36].
Inhibition of endogenous VEGF by topical application
of anti-VEGF antibody in the ischemic cortex de-
creased the blood-brain barrier (BBB) disruption [37].
VEGF is in part responsible for the BBB disruption
during the early stage of focal cerebral ischemia [37] .
These adverse effects suggested that caution should
be heeded when considering the use of only VEGF for
stroke patients [38].
EPO is a pleiotropic factor [39] . EP O and EPO
receptor (EP O -R) are expressed in neurons, astrocytes,
and endothelial cells after focal permanent ischemia
in mice. EPO is a neuroprotective factor [40-41], which
improves functional recovery and reduces neuronal
apoptosis [42] and inflammation [43]. EPO has a mi-
togenic and positive chemotactic effect on endothelial
cells and endothelial progenitor cells [44-45]. It sti-
mulates angiogenesis in vitro and i n v i v o [18,40].
Therefore, the EPO/EP O -R system is implicated in
the process of neuroprotection [4 6] and restructuring
(such as angiogenesis) after ischemia [18,46]. In this
study, catalpol increased EPO expression in neurons
and endothelial-like cells surrounding the vessels.
Therefore, catalpol may improve neurofunction and
neural and vascular remodeling after stroke by acti-
vating EPO .
In this study, we observed that catalpol in-
creased VEGF expression but did not increase vascu-
lar permeability (Fig 3b), which may be related to the
simultaneous increase of EPO expression in the brain.
EPO reduces the side effects of VEGF, which protects
the BBB against VEGF-induced permeability in vitro
[19]. EPO and VEGF promote neural outgrowth
(Bocker-Meffert et al., 2002) and exhibit equal angi-
ogenic potential [18]. Furthermore VEGF modulates
erythropoiesis through regulation of adult hepatic
erythropoietin synthesis [ 47], a n d EPO -R promotes
V EG F e xp re ssion and angiogenesis in peripheral
ischemia in mice [48]. Therefore, the advantageous
reciprocal interactions between EPO and VEGF on
angiogenesis, which can be induced by catapol may
be an effective way to treat stroke patients. Brain in -
j u r y h e l p s E PO to cross the BBB [49-50], w h i c h m a y
then produce a synergistic effect with VEGF. Catalpol
is the effective component of Rehmannia glutinosa,
which can increase blood level o f EPO (data not
shown). Moreover, catapol can cross the BBB, as de-
tected by HPLC even in normal rats [51].
In conclusion, our data suggested that catalpol
modul ated angiogenesis through increased EPO and
VEGF after stroke, which may be the mechanism by
which catalpol reduced ischemic neuronal damage
and enhanced functional recovery. Taken to ge t he r ,
these data suggested that catalpol may improve col-
lateral circulation and pr o vi d e impact on stroke pa-
tients through new blood vessel formation. Future
research may elucidate the specific signaling path-
ways through which catalpol increases angioge n es i s.
Acknowledgements
This work was supported by grants from the
Fundamental Research Funds for the Central Univer-
sities (No.XDJK2009C081) and Southwest University
Dr. Foundation (No.104290-20710906) and N SF C
General Projects (No.81073084).
Conflict of Interests
The authors have declared that no conflict of in-
terest exists.
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