Carvedilol prevents cardiac hypertrophy and overexpression
of hypoxia-inducible factor-1a and vascular endothelial growth
factor in pressure-overloaded rat heart
Kou-Gi Shyua,b, Jer-Young Liouc, Bao-Wei Wanga, Wei-Jen Fang a, Hang Changd,*
aDepartment of Education and Research, Shin Kong Wu Ho-Su Memorial Hospital, 95 Wen-Chang Rd,
Taipei, Taiwan; bGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan;
cDepartment of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan; dTaipei City
Hospital, Taipei, Taiwan
Received 04 May 2004; accepted in revised form 14 January 2005

2005 National Science Council, Taipei
Key words: aortic banding, cardiac hypertrophy, HIF-1a, pressure overload, VEGF
Summary
The use of b-blockers has emerged as a beneficial treatment for cardiac hypertrophy. Hypoxia-inducible
factor-1a (HIF-1a) is tightly regulated in the ventricular myocardium. However, the expression of HIF-1a
in cardiac hypertrophy due to pressure overload and after treatment with b-blocker is little known. To
evaluate the effect of carvedilol on both myocardial HIF-1a expression and cardiac hypertrophy, infra-
renal aortic banding was performed for 4 weeks in adult Sprague-Dawley rats to induce cardiac hyper-
trophy. Carvedilol at 50 mg/kg body weight per day after surgery was given. Heart weight and the ratio of
heart weight and body weight increased significantly after aortic banding for 4 weeks in the absence of drug
treatment. Mean arterial pressure increased from 80 ± 9 mmHg in the sham group to 94 ±5 mmHg
(p < 0.001) in the banding group. Echocardiography showed concentric hypertrophy after aortic banding.
Mean arterial pressure decreased after treatment with carvedilol. The increased wall thickness and heart
weight was reversed to normal by carvedilol. Western blot showed that HIF-1a, vascular endothelial
growth factor (VEGF) and brain natriuretic peptide (BNP) proteins were up-regulated and nerve growth
factor-b (NGF-b) down-regulated in the banding group. Treatment with valsartan, doxazosin, or
N-acetylcysteine did not significantly affect HIF-1a and VEGF proteins expression in the banding groups.
Real-time polymerase chain reaction showed that mRNA of HIF-1a, VEGF and BNP increased and
mRNA of NGF-b decreased in the banding group. Treatment with carvedilol reversed both protein and
mRNA of HIF-1a, VEGF, BNP, and NGF-b to the baseline values. Increased immunohistochemical
labeling of HIF-1a, VEGF, and BNP in the ventricular myocardium was observed in the banding group
and carvedilol again normalized the labeling. In conclusion, HIF-1a, VEGF, and BNP mRNA and protein
expression were up-regulated, while NGF-b mRNA and protein was downregulated in the rat model of
pressure-overloaded cardiac hypertrophy. Treatment with carvedilol is associated with a reversal of
abnormal regulation of HIF-1a,VEGF, BNP, and NGF-b in the hypertrophic myocardium.
Cardiac hypertrophy is an ultimately maladaptive
response to pathological status of increased hemo-
dynamic overload leading to cardiovascular mor-
bidity and mortality. Chronic cardiac hypertrophy
may be deleterious because it increases the risk for
the development of heart failure and premature
death [1]. Cardiac hypertrophy is usually accom-
panied by complex changes in gene reprogram-
ming [2]. These changes include genes that modify
*To whom correspondence should be addressed. Fax +886-2-
2836-5775; E-mail: T002558@ms.skh.org.tw
Journal of Biomedical Science 12: 409–420, 2005. 409
DOI 10.1007/s11373-005-3008-x

energy metabolism and encode components of
hormonal pathways.
Hypoxia-inducible factor 1 (HIF-1), a tran-
scription factor, is now recognized to be a key
physiologic regulator of gene expression that
responds to changes in cellular oxygen tension [3].
HIF-1 is a heterodimeric DNA complex composed
of HIF-1a and HIF-1b.Under hypoxic conditions,
both HIF-1a protein levels and activity of the HIF-
1a transactivation domains increases [4, 5]. HIF-1a
is involved in energy metabolism. In the progres-
sion of heart failure, in which impaired energy
metabolism may occur, HIF-1a is likely to be
involved in the activation of the glycolytic system
[6, 7]. In addition to the hypoxic pathway mediat-
ing HIF-1a expression, other nonhypoxic path-
ways factors such as neurohormonal activators and
mechanical stress have also been shown to induce
HIF-1a expression in vascular smooth muscle cells
[8, 9]. Recently Kim et al. [10] demonstrated that
acute hemodynamic overload induced early expres-
sion of HIF-1a and vascular endothelial growth
factor (VEGF) in rat myocardium.
The expression of HIF-1a in cardiac hyper-
trophy is little known. Tissue ischemia has been
reported in congestive heart failure [11], possibly
due to a combination of increased wall stress and
coronary vasoconstriction and endothelial dys-
function. Increased wall stress occurs in hyper-
trophic myocardium. HIF-1a may play a role in
the hypertrophic myocardium because of pres-
sure overload. The use of b-blockers has emerged
as a beneficial treatment in hypertensive patients
with increased left ventricular mass [12]. How-
ever, the link of gene expression to b-blocker
treatment in the hypertrophic heart is rarely
reported. Accordingly, we sought to evaluate the
effect of carvedilol on both myocardial HIF-1a
expression and cardiac hypertrophy in a rat
model of pressure overload induced by abdom-
inal aortic constriction.
Methods
Rat model of abdominal aortic constriction
On the day of surgery, Adult Sprague-Dawley rats
weighing 250–300 g were anesthetized with pento-
barbital sodium (80 mg/kg) and the aorta was
exposed via abdominal midline incision. Rats were
randomly divided into four groups: (1) sham-
operated, (2) sham-operated and treatment with
carvedilol, (3) pressure-overloaded (aortic band-
ing), and (4) pressured-overloaded and treatment
with carvedilol rats. A polyethylene catheter
(PE10) was put on the surface of abdominal aorta
distal to the renal arteries. Then the catheter and
aorta were tightly constricted with 6-0 silk. Sham-
operated control animals were prepared in a
similar manner, except that the aorta was not
constricted. After the procedure, the catheter was
fully withdrawn and the abdominal wound was
sutured and let the rats recover. In the treatment
group, the rat was given 50 mg/kg body weight per
day of carvedilol in drinking water after surgery
for 4 weeks. The perioperative mortality in the
aortic banding group was around 10%. In addition
to carvedilol, valsartan (a selective angiotenisn II
receptor antagonist) at 30 mg/kg body weight per
day, doxazosin (a-adrenergic receptor antagonist)
at 3 mg/kg body weight per day, and N-acetylcys-
teine (antioxidant) at 250 mg/kg body weight per
day were also given to different banding rats. All
animal procedures were performed in accordance
with institutional guideline and conformed with
the Guide for the Care and Use of Laboratory
Animals as published by the US NIH.
Hemodynamic monitor
Four weeks after the surgery, the animals were
anesthetized with pentobarbital sodium (80 mg/
kg), and their carotid arteries were cannulated
with polyethylene catheters to measure mean
arterial pressure. Heart rate was measured
through a Grass model tachograph preamplifier.
After the measurement of hemodynamic data, the
rats were sacrificed to remove the heart to
perform the following experiments. Each hemo-
dyamic data was presented as a mean from three
measurements.
Assessment of cardiac hypertrophy and function
Cardiac function of aortic banding rats was
evaluated noninvasively by echocardiography per-
formed with an Acuson Sequoia 512 machine
using a 15-MHz probe at the day of sacrifice,
4 weeks after surgery. Left ventricular percent
fractional shortening, left ventricular end-diastolic
dimension, left ventricular end-systolic dimension,
410

interventricular septum thickness, and left ventric-
ular posterior wall thickness were calculated.
The sonographer was blinded to the randomization
of rats. The following experiments were performed
by a technician blinded to the design of the study.
Western blot analysis
Tissue samples from left ventricle were homoge-
nized in modified RIPA buffer (50 mmol/l tris
[pH 7.4], 1% IGEPAL CA-630 (Sigma), 0.25%
sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/
l EDTA, 1 mmol/l phenylmethylsulfonyl fluoride,
and 1 lg/ml aprotinin, leupetin, and pepstatin).
Nuclear and cytosolic protein samples were mixed
with sample buffer, boiled for 10 min, separated
by SDS-PAGE under denaturing conditions, and
electroblotted to nitrocellulose membranes. The
nitrocellulose membranes were blocked by inc-
ubation in blocking buffer, incubated with anti-
VEGF, or anti-HIF-1a, anti-BNP, or anti-NGF-b
antibody, washed, and incubated with horseradish
peroxidase-conjugated secondary antibody. Signals
were visualized by chemiluminenescent detection.
RNA isolation and reverse transcription
Total RNA was isolated from frozen left
ventricle using the single-step acid guanidinium
thiocyanate/phenol/chloroform extraction meth-
od. Total RNA (1 lg) was incubated with 200 U
of Moloney–Murine Leukemia Virus reverse
transcriptase in a buffer containing a final con-
centration of 50 mmol/l Tris–Cl (pH 8.3),
75 mmol/l KCl, 3 mmol/MgCl2, 20 U of RNase
inhibitor, 1 lmol/l polydT oligomer, and
0.5 mmol/l of each dNTP in a final volume of
20 ll. The reaction mixture was incubated at 42 C
for 1 h and then at 94 C for 5 min to inactivate
the enzyme. A total of 80 ll of diethyl pyrocar-
bonate treated water was added to the reaction
mixture before storage at )70 C.
Real-time PCR
A Lightcycler (Roche Diagnostics, Mannheim,
Germany) was used for real-time PCR. cDNA
was diluted 1 in 10 with nuclease-free water. 2 ll
of the solution was used for the Lightcycler
SYBR-Green mastermix (Roche Diagnostics):
0.5 lmol/l primer, 5 mmol/l magnesium chloride,
and 2 ll Master SYBR-Green in nuclease-free
water in a final volume of 20 ll. The primers used
for VEGF, HIF-1a, NGF-b, BNP, and GAPDH
were: forward, 5¢-CACCCACGACAGAAGG-3¢,
5¢-AG TCGGACAGCCTCAC-3¢, 5¢-AACCAAT-
AGCT GCCCG-3¢, 5¢-CTCAAAGGACCAAGGC-
3¢, and 5¢-CATCACCATCTTCCAGGAGC-3¢,
respectively; reverse 5¢-TCACAGTGAACGCTC
CC-3¢, 5¢-TGCTGCCTTGTATGGGA-3¢, 5¢-CG
TCTGTTGTCAACGCC-3¢, 5¢-GTCGGTAAGG
TAGAGGC-3¢, and 5¢-GGATGATGTTCTGGG
CTGCC-3¢, respectively. The initial denaturation
phase for rat VEGF was 5 min at 95 C followed
by an amplification phase as detailed below:
denaturation at 95 C for 10 s; annealing at
55 C for 10 s; elongation at 72 C for 15 s;
detection at 79 C and for 37 cycles. The ampli-
fication phase program of rat HIF1a as detailed
below: denaturation at 95 C for 10 s; annealing at
57 C for 10 s; elongation at 72 C for 15 s;
detection at 80 C and for 36 cycles. The ampli-
cation phase for rat BNP was denaturation at
95 C for 5 s; annealing at 60 C for 3 s; elonga-
tion at 72 C for 5 s; detection at 72 C and for 40
cycles. The amplication phase for rat NGF-b was
denaturation at 95 C for 10 s; annealing at 56 C
for 5 s; elongation at 72 C for 20 s; detection at
72 C and for 39 cycles. Amplification, fluores-
cence detection, and post-processing calculation
were performed using the Lightcycler apparatus.
Individual PCR product was analyzed for DNA
sequence to confirm the purity of the product. The
size of the PCR products for VEGF, HIF-1a,
NGF-b, BNP, and GAPDH were 421, 436, 474,
294, and 405 bp, respectively.
Immunohistochemistry
Slides were dried overnight at room temperature.
Snap-frozen sections were postfixed in 4% para-
formaldehyde for 20 min, treated in 3% hydrogen
peroxide/PBS for 25 min, blocked in 5% normal
rabbit serum for 20 min, blocked with biotin/
avidin for 15 min each, and incubated with the
following: primary antibody (anti-VEGF, anti-
HIF-1a, anti-BNP, or anti-NGF-b antibody) for
2 h at room temperature, biotinylated rabbit-anti
mouse IgG at 1:400 for 30 min, and Vector Elite
ABC biotin-avidin-peroxidase complex for 30 min;
411

sections were then developed with diaminobenzi-
dine and diaminobenzidine enhancer (Vector),
counterstained with hematoxylin, and mounted.
Statistical analysis
All results were expressed as mean ± SD. Statis-
tical significance was evaluated using analysis of
variance followed by Tukey-Kramer multiple
comparisons test (GraphPad Software Inc., Dan
Diego, CA). A value of p < 0.05 was considered
to denote statistical significance.
Results
Hemodynamic and echocardiographic change after
banding and treatment with carvedilol
The mean of baseline body weight was 270 gm. The
mean of baseline thickness of interventricular sep-
tum and left ventricular posterior wall was 1.1 mm
and 1.3 mm, respectively. The baseline body weight
and echocardiographic parameters were similar in
each group (n = 36 for all groups). As shown in
Table 1, heart rate increased significantly after
aortic banding for 4 weeks from 329 ± 8 bpm in
the sham group to 352 ± 10 bpm (p < 0.001) in
the banding group.Meanarterial pressure increased
from 80 ± 9 mmHg in the sham group to
94 ± 5 mmHg (p < 0.001) in the banding group.
Echocardiography showed that the thickness of
interventricular septum and left ventricular poster-
ior wall also increased significantly after aortic
banding. Treatment with carvedilol significantly
reduced the heart weight and the ratio of heart
weight andbodyweight.Mean arterial pressure also
decreased after treatment with carvedilol. The
increased wall thickness was reversed to normal by
carvedilol. Treatmentwith carvedilol in the banding
and sham groups significantly decreased heart rate
as compared with both groups without treatment.
A dose-dependent effect of carvedilol on wall
thickness was observed. As shown in Table 2, lower
dose of carvedilol at 2.5 mg/kg/day showed less
effect on reversed wall thickness and reduction in
mean arterial blood pressure than higher dose of
carvedilol at 12.5 mg/kg/day and 25 mg/kg/day.
Treatment with propranolol at 30 mg/kg/day
showed similar effect on hemodyanmic and echo-
cardiographic change as compared to treatment
with carvedilol at 12.5 mg/kg/day (data not shown).
As shown in Table 3, treatment with valsartan and
doxazosin also decreased heart weight, mean arte-
rial pressure, and thickness of interventricular
septum after banding. N-acetylcysteine did not
affect the heart weight, mean arterial pressure, and
echocardiographic parameters after banding.
Western blot analysis after aortic banding and
treatment with carvedilol
To evaluate the effect of aortic banding and
cavedilol on the HIF-1a and VEGF protein
expression, Western blotting was performed. The
Table 1. Hemodynamic and echocardiographic parameters at the end of study.
Sham Banding Sham/C Banding/C
N 9 10 9 8
Body weight, g 287 ± 5 279 ± 9 277 ± 4 274 ± 9
Heart weight, mg 688 ± 40 892 ± 50*,** 665 ± 40 740 ± 34
Heart weight/body weight, mg/g 2.4 ± 0.2 3.2 ± 0.1*,** 2.4 ± 0.1 2.7 ± 0.1
Heart rate, min)1 329 ± 8 352 ± 10*,** 302 ± 7*** 325 ± 12
MAP, mmHg 80 ± 9 94 ± 5*,** 71 ± 8 79 ± 6
IVSTd, mm 1.2 ± 0.1 2.2 ± 0.1*,** 1.1 ± 0.1 1.5 ± 0.1
LVPWT, mm 1.2 ± 0.3 2.2 ± 0.1*,** 1.3 ± 0.2 1.5 ± 0.1
LVEDD, mm 6.3 ± 0.9 6.5 ± 0.8 6.4 ± 0.4 6.1 ± 1.0
LVESD, mm 3.3 ± 0.7 3.8 ± 0.3 3.4 ± 0.2 3.1 ± 0.2
FS, % 48 ± 4 45 ± 4 46 ± 5 49 ± 2
LV mass, mg 656 ± 60 1303 ± 70*,** 652 ± 49 761 ± 52
Data are means ± SD. C, carvedilol treated at 50 mg/kg/day; MAP, mean arterial pressure; IVSTd, interventricular septum thickness;
LVPWT, left ventricular posterior wall thickness; LVEDD, left ventricular end-diastolic dimension; LVESD, left ventricular end-
systolic dimension; FS, fraction shortening. *p < 0.001 vs. sham group. **p < 0.001 vs. Banding/C group.
412

HIF-1a protein increased 2.1-fold at 4 weeks of
induction of aortic banding as compared to sham
group (p < 0.001). VEGF protein also increased
2.4-fold at 4 weeks of induction of aortic banding
as compared to sham group (Figure 1). Treatment
with carvedilol in the banding group significantly
blocked the increase of HIF-1a and VEGF pro-
teins-induced by aortic banding. Aortic banding
significantly increased BNP protein expression
(2.7-fold, p < 0.001 vs. sham group) and de-
creased NGF-b protein expression (0.8-fold,
p < 0.05 vs. sham group). Treatment with carv-
edilol in the banding group reversed the BNP and
NGF-b protein expression to the baseline levels.
Treatment with carvedilol in the sham group did
not affect the protein expression of HIF-1a,
VEGF, BNP, and NGF-b. Treatment with
carvedilol at 2.5 mg/kg/day did not block the
increase of HIF-1a and VEGF proteins-induced
by aortic banding. Treatment with carvedilol at
12.5 mg/kg/day in the banding group significantly
blocked the increase of HIF-1a (from 2.1 ± 0.1-
fold to 1.5 ± 0.1-fold; p < 0.05, n = 3) and
VEGF proteins-induced (from 2.4 ± 0.2-fold to
1.6 ± 0.1-fold; p < 0.05, n = 3) by aortic band-
ing. Treatment with propranolol at 30 mg/kg/day
in the banding group also significantly blocked the
increase of HIF-1a and VEGF proteins-induced
by aortic banding (data not shown). Valsartan and
doxazosin reduced the HIF-1a and VEGF pro-
teins-induced by aortic banding, but the difference
did not reach statistical significance as compared
Table 2. Hemodynamic and echocardiographic parameters at the end of study.
Banding/C2.5 Banding/C12.5 Banding/C25
N 6 8 6
Body weight, g 295 ± 10 274 ± 9** 281 ± 7*
Heart weight, mg 826 ± 26 740 ± 34*** 758 ± 30**
Heart weight/body weight, mg/g 2.8 ± 0.2 2.7 ± 0.1 2.7 ± 0.2
Heart rate, min)1 332 ± 6 325 ± 12 324 ± 7
MAP, mmHg 94 ± 5 79 ± 6*** 78 ± 6***
IVSTd, mm 1.8 ± 0.3 1.5 ± 0.1* 1.4 ± 0.2***
LVPWT, mm 2.0 ± 0. 1.5 ± 0.1*** 1.6 ± 0.1***
LVEDD, mm 6.4 ± 1.0 6.1 ± 1.0 6.2 ± 0.9
LVESD, mm 3.5 ± 0.7 3.1 ± 0.2 3.3 ± 0.8
FS, % 46 ± 5 49 ± 2 47 ± 3
Data are means ± SD. C2.5, C12.5, and C25, carvedilol treated at 2.5 mg/kg/day, 12.5 mg/kg/day, and 25 mg/kg/day, respectively;
Other abbreviations as the same in Table 1. *p < 0.05 vs. Banding/C2.5. **p < 0.01 vs. Banding/C2.5. ***p < 0.001 vs. Banding/
C2.5.
Table 3. Hemodynamic and echocardiographic parameters at the end of study.
Banding Banding/V Banding/Do Banding/NAC
N 8 7 6 6
Body weight, g 285 ± 9 280 ± 5 290 ± 9 293 ± 8
Heart weight, mg 912 ± 30 757 ± 13*** 784 ± 24*** 908 ± 25
Heart weight/body weight, mg/g 3.2 ± 0.2 2.7 ± 0.4* 2.7 ± 0.3* 3.1 ± 0.3
Heart rate, min)1 346 ± 9 326 ± 6** 332 ± 9* 338 ± 11
MAP, mmHg 98 ± 7 86 ± 7* 79 ± 9*** 97 ± 8
IVSTd, mm 2.1 ± 0.1 1.5 ± 0.1*** 1.7 ± 0.2*** 2.0 ± 0.2
LVPWT, mm 2.2 ± 0.2 1.6 ± 0.2*** 1.8 ± 0.2* 2.1 ± 0.4
LVEDD, mm 6.5 ± 0.8 6.2 ± 0.9 6.3 ± 0.8 6.5 ± 0.8
LVESD, mm 3.8 ± 0.3 3.2 ± 0.9 3.2 ± 0.8 3.6 ± 0.9
FS, % 45 ± 4 48 ± 7 48 ± 7 45 ± 6
Data are means ± SD. V, varsartan treated; Do, doxaben treated; N, N-acetylcysteine treated; Other abbreviations as the same in
Table 1. *p < 0.05 vs. banding group. **p < 0.01 vs. banding group. ***p < 0.001 vs. banding group.
413

to the banding group without treatment
(Figure 2). However, treatment with both drugs
significantly blocked the increase of BNP protein-
induced by aortic banding. N-acetylcysteine did
not affect any of the four proteins after aortic
banding (Figure 3).
Expression of mRNA by real-time PCR after aortic
banding and treatment with carvedilol
We used real-time PCR to evaluate the effect of
aortic banding and cavedilol on the HIF-1a and
VEGF mRNA levels. As shown in Figure 4,
HIF-1a mRNA increased 1.9-fold at 4 weeks of
induction of aortic banding as compared to the
sham group (p < 0.001). VEGF mRNA also
increased 2-fold at 4 weeks of induction of aortic
banding as compared to sham group
(p < 0.001). Treatment with carvedilol in the
banding group significantly inhibited the increase
of HIF-1a and VEGF mRNA-induced by aortic
banding. As shown in Figure 5, aortic banding
significantly increased BNP mRNA expression
(1.8-fold, p < 0.001 vs. sham group) and
Figure 1. Effect of aortic banding and treatment with carvedilol (c) on protein expression. (a) Representative Western blot for
hypoxia-inducible factor 1a (HIF-1a), vascular endothelial growth factor (VEGF), nerve growth factor-b (NGF-b), and brain
natriuretic peptide (BNP) after induction of aortic banding for 4 weeks with or without treatment with cardvedilol. (b) Quantita-
tive analysis of HIF-1a,VEGF, NGF-b, and BNP protein levels after induction of aortic banding for 4 weeks with or without
treatment with cardvedilol. The values from experiment groups have been normalized to values in sham group. Equal loading of
samples was verified by staining with GAPDH-specific monoclonal antibody. n = 4–5 in each group.
414

decreased NGF-b mRNA expression (0.7-fold,
p < 0.05 vs. sham group). Treatment with carv-
edilol in the banding group completely blocked
the increase of BNP and NGF-b mRNA expres-
sion-induced by aortic banding. Treatment with
carvedilol in the sham group did not affect the
Figure 2. Effect of valsartan and doxaben on proteins expression. (a) Representative Western blot for hypoxia-inducible factor 1a
(HIF-1a), vascular endothelial growth factor (VEGF), nerve growth factor-b (NGF-b), and brain natriuretic peptide (BNP) after
induction of aortic banding for 4 weeks with or without treatment with valsartan (V) or doxaben (Do). (b) Quantitative analysis of
HIF-1a, VEGF, NGF-b, and BNP protein levels after induction of aortic banding for 4 weeks with or without treatment with val-
sartan (V) or doxaben (Do). n = 6–7 in each group.
415

mRNA expression of HIF-1a, VEGF, BNP, and
NGF-b.
Increased immunohistochemical labeling of HIF-1a
and VEGF after aortic banding
Immunohistochemical stain confirmed the previ-
ous findings from Western blots and real-time
PCR. Increased labeling of HIF-1a, VEGF and
BNP in the ventricular myocardium was observed
after induction of aortic banding for 4 weeks
(Figure 6). Treatment with carvedilol in the band-
ing group decreased the immunohistochemical
labeling of HIF-1a, VEGF, and BNP. Decreased
immunohistochemical labeling of NGF-b in the
ventricular myocardium was observed after induc-
tion of aortic banding for 4 weeks. Treatment with
carvedilol in the banding group increased the
immunohistochemical labeling of NGF-b. Treat-
ment with carvedilol in the sham group did not
affect the immunohistochemical labeling of HIF-
1a, VEGF, BNP, and NGF-b (data not shown).
Discussion
In this study, we demonstrated that both HIF-1a
and VEGF mRNA and protein expression were
up-regulated in a rat model of pressure overload-
induced cardiac hypertrophy. The model of car-
diac hypertrophy was confirmed by morphological
and gene profile study. Blood pressure and heart
weight increased after aortic banding. Cardiac
echocardiography showed concentric hypertrophy
Figure 3. Representative Western blot for hypoxia-inducible
factor 1a (HIF-1a), vascular endothelial growth factor
(VEGF), nerve growth factor-b (NGF-b), and brain natri-
uretic peptide (BNP) after induction of aortic banding for
4 weeks with or without treatment with N-acetylcysteine
(NAC). Similar results were found in another two experi-
ments.
Figure 4. Effect of aortic banding and treatment with carvedilol (c) on HIF-1a and VEGF mRNA expression. Upper panel, repre-
sentative real time polymerase chain reaction for VEGF (a) and HIF-1a (b) after induction of aortic banding for 4 weeks with or
without treatment with cardvedilol; lower panel, fold increases in VEGF (a) and HIF-1a (b) mRNA as a result of induction of
aortic banding for 4 weeks with or without treatment with cardvedilol. The values from experiment groups have been normalized
to matched GAPDH measurement and then expressed as a ratio of normalized values to mRNA in sham group. NC indicates neg-
ative control. n = 4–5 in each group.
416

of left ventricle after aortic banding for 4 weeks.
The hemodynamic and echocardiographic data
that are presented certainly support a pressure
overload state. Cardiac BNP mRNA and protein
expression were measured to confirm the presence
of cardiac hypertrophy. Expression of BNP gene is
one of the most reliable markers for activation of
the hypertrophic program in clinical states and
experimental models associated with hypertrophy
[13]. The elevated BNP supports the presence of
cardiac hypertrophy.
Although expressions of other genes such as
natriuretic peptide have been extensively examined
in cardiac hypertrophy, much less is known about
HIF-1a [13, 14]. Aortic banding is known to
increase concentrations of several humoral factors
including angiotensin II, epinephrine, endothelin-1
as well as blood pressure [15, 16]. The presence of
circulating humoral factors secondary to pressure
overload that can trigger the cardiac hypertrophic
response and alter gene expression. Reactive oxy-
gen species also play a key role in pressure
overload-induced cardiac hypertrophy and anti-
oxidant attenuates myocardial hypertrophy
[17, 18]. Recent studies suggest that HIF-1a
mRNA and protein can be induced under norm-
oxic conditions by growth factors and hormones in
vascular smooth muscle cells [8, 9] and by
mechanical stress in rat myocardium [10].
Although increased wall stress and myocardial
stretching might be responsible, humoral factors
secondary to mechanical load could also contrib-
ute to the upregulation of HIF-1a mRNA and
protein expression in the model of pressure over-
load-induced cardiac hypertrophy. In the model of
pressure overload, the blood pressure and left
ventricular mass were increased and cytokine
released due to neurohormonal activation.
Accordingly, HIF-1a and VEGF were up-regu-
lated in the pressure overload cardiac hypertro-
phy. Carvedilol treatment decreased blood
pressure and left ventricular mass and may also
decrease neurohormal activation. Therefore, carv-
edilol treatment reverses the abnormal expression
of HIF-1a and VEGF in the hypertrophic myo-
cardium.
The VEGF expression in hypertrophic
myocardium is also little known. The VEGF
Figure 5. Effect of aortic banding and treatment with carvedilol (c) on NGF-b and BNP mRNA expression. Upper panel, repre-
sentative real time polymerase chain reaction for NGF-b (a) and BNP (b) after induction of aortic banding for 4 weeks with or
without treatment with cardvedilol; lower panel, fold increases in NGF-b (a) and BNP (b) mRNA as a result of induction of aortic
banding for 4 weeks with or without treatment with cardvedilol. The values from experiment groups have been normalized to mat-
ched GAPDH measurement and then expressed as a ratio of normalized values to mRNA in sham group. NC indicates negative
control. n = 4–5 in each group.
417

expression in heart failure is controversial. Tham
et al. reported up-regulation of VEGF-A without
angiogenesis in a mouse model of dilated cardio-
myopathy caused by mitochondrial dysfunction
[19]. However, Abraham et al. demonstrated that
VEGF-A was down-regulated and capillary
density was decreased in patients with dilated
cardiomyopathy [20]. In patients with congestive
heart failure, the blood levels of VEGF were also
inconsistent [21, 22]. Decreased NGF-b and
elevated BNP in our study may indicate that
asymptomatic heart failure in our model of
pressure-overloaded cardiac hypertrophy. In this
study, we demonstrated that HIF-1a mRNA and
protein expression was upregulated in a pressure
overload-induced cardiac hypertrophy. The in-
crease in HIF-1a protein will increase the expres-
sion of its target gene, VEGF.
Use of b-blocker to treat patients with hyper-
tension or congestive heart failure is beneficial.
The functional recovery resulting from b-blocker-
therapy has been shown to restore the unfavorable
gene expression that control Ca2+ handling in the
failing heart [23, 24]. Whether b-blocker therapy
can alter HIF-1a, a major physiologic regulator of
many genes, in myocardium is not known in
cardiac hypertrophy. Change gene expression after
b-blocker therapy in human was obtained from
right ventricle [23, 25]. However gene expression of
the left ventricle is not necessarily the same as in
the right ventricle.
Carvedilol is a nonselective, vasodilating
b-blocker with potent antioxidant and free rad-
ical-scavenging properties that is used in the
treatment of hypertension, angina, and congestive
heart failure [26]. In the present study, carvedilol
repressed cardiac hypertrophy induced by aortic
banding which is consistent with previous study
reported by Massart et al. [16]. In this study, both
angiotensin II receptor antagonist and a-adren-
ergic receptor antagonist decreased blood pres-
sure induced by aortic banding, but the HIF-1a
and VEGF proteins did not change significantly.
These data implicated that change of HIF-1a and
VEGF gene expression by carvedilol was not con-
tributed by hemodynamic alteration. Antioxidant
Figure 6. Immunohistoichemical staining of left ventricular myocardium after induction of aortic banding for 4 weeks with or
without treatment with cardvedilol (c).
418

with N-acetylcysteine did not alter HIF-1a and
VEGF proteins, implying the negligible effect of
oxidative stress changes on this phenomenon.
Other b-receptor blockades such as propranolol
have similar effect on change of HIF-1a and
VEGF gene expression induced by aortic band-
ing. The potential mechanism that carvedilol
reverses HIF-1a and VEGF gene expression in
pressure-overloaded heart is through b-receptor-
blockade effect. Our study proves that there is a
class effect of b-receptor blockade on change of
HIF-1a and VEGF gene expression induced by
aortic banding.
Cardiac hypertrophy can be induced in many
hypertensive animal models. The classic animal
models include stroke prone spontaneously
hypertensive rats fed with a high-salt-diet, uni-
nephrectomized rats receiving deoxycorticoster-
one acetate and a high-salt diet, renovascular
hypertensive rats, and rats infused with angioten-
sin II or norepinephrine. The mechanisms of
cardiac hypertrophy induced by these models
may not be similar to that of our aortic banding
model. It needs further study to investigate
whether the effect of carvedilol applies to these
hypertensive animal models.
Reducing heart rate has been reported to
increase myocardial VEGF production [25]. In
our study, carvedilol reduced heart rate both in the
treated sham and banding groups. However,
VEGF and HIF-1a mRNA and protein expression
in the treated sham group were similar to that in
the sham group without treatment. This finding
indicated that the up-regulation of VEGF and
HIF-1a mRNA and protein expression in the
pressure overload-induced cardiac hypertrophy
was not due to bradycardia effect. However, we
cannot exclude the possibility that hemodynamic
change after carvedilol treatment may alter the
HIF-1a and VEGF expression in our study.
In summary, HIF-1a and VEGF mRNA and
protein expression were up-regulated in the rat
model of cardiac hypertrophy because of pressure
overload. Treatment with carvedilol is associated
with a reversal of abnormal regulation of HIF-1a
and VEGF in the hypertrophic myocardium.
These findings emphasize the potential implica-
tions of carvedilol on pressure-overload induced
cardiac hypertrophy to clinical setting such as
hypertensive patients with increased left ventricu-
lar mass.
Acknowledgments
This study was supported in part from National
Science Council, Taiwan.
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