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Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
www.elsevier.com/locate/yjmcc
Original Article
Angiogenic signal triggered by ischemic stress induces myocardial
repair in rat during chronic infarction
Shoji Fukuda a, Shigeaki Kaga a, Hiroaki Sasaki a, Lijun Zhan a, Li Zhu a, Hajime Otani b,
Reni Kalfin c, Dipak K Das, Nilanjana Maulik a,*
b
a
Molecular Cardiology Laboratory, University of Connecticut Medical School, Farmington, Connecticut, USA
Department of Thoracic and Cardiovascular Surgery, Kansai Medical University, School of Medicine, Osaka, Japan
c
Bulgarian Academy of Sciences, Institute of Physiology, Sofia, Bulgaria
Received 5 November 2003; received in revised form 23 January 2004; accepted 4 February 2004
Abstract
In the present study, we examined a novel method of stimulating myocardial angiogenesis through ischemic preconditioning (IP) in the
form of in vivo four repetitive cycles of coronary artery occlusion each followed by reperfusion. Rats divided into 4 groups: Control+Sham
surgery (CS), Control+ Left anterior descending coronary artery (LAD) occlusion (CMI), IP+ Sham surgery (IPS) and IP+LAD occlusion
(IPMI). For cardiac function, rats were subjected to stress testing with dobutamine after 2, 4, 7, 14 and 21 operative days. Capillary density
(CD) and arteriolar density (AD) were evaluated by immunohistochemistry. Western blot was performed to examine the expression pattern for
VEGF and anti-death candidates, Bcl-2 and survivin. Blood flow and the extent of endothelial and cardiomyocyte cell death were examined.
The protein/DNA array was performed to determine the status of various transcription factor related to stress signal. Left ventricular functional
reserve was better preserved in IPMI compared to the CMI group. The infarct size and apoptotic cell death were reduced in IPMI group
significantly. Left ventricular regional blood flow, perfused capillary density and AD increased significantly in the IPMI group. VEGF, Bcl-2
and survivin expression were increased in IPMI compared to CMI. VEGF mediated vascular permeability was controlled in the IPMI due to
suppression of c-Src in the infarcted myocardium. Our study documented first time the ability of IP to induce angiogenesis in the infarcted
myocardium along with the activation of several transcription factors such as Stat3, Pax-5, NFjB, TFIID, SP1 and reduction of VEGF
mediated vascular permeability by inhibition of c-Src in IPMI group thereby reducing ischemic injury in rat MI model.
© 2004 Elsevier Ltd. All rights reserved.
Keywords: Angiogenesis; Ischemic Stress; Myocardial Infarction; Edema; VEGF
1. Introduction
Angiogenic therapy for the human heart is currently being
vigorously pursued. In the past ten years, alternative
revascularization/angiogenesis strategies have progressed
from bench to bedside, focusing on the capillary sprouting
and/or growth of new vessels to replace the old.
However, most of the strategies involve the delivery of
growth factors. Very little success with these strategies has
been demonstrated so far for various reasons. Our approach
to IP mediated angiogenesis and growth factors/receptors
* Correspondence: Nilanjana Maulik, Ph. D. Molecular Cardiology
Laboratory, Cardiovascular Division, Department of Surgery University of
Connecticut School of Medicine, Farmington, CT 06030-1110, USA Tel:
(860) 679-2857 Fax: (860) 679-2825.
E-mail address: [email protected] (N. Maulik).
© 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.yjmcc.2004.02.002
stimulation for cardioprotection is a very novel and potentially very exciting strategy for myocardial protection. Preconditioning of heart by repeated ischemia and reperfusion
was found to delay the onset of subsequent irreversible ischemic injury [1]. It is well known that preconditioning
provides cardioprotection by reducing subsequent postischemic ventricular dysfunction, decreasing incidence of
arrhythmias, and infarct size. Such myocardial preservation
by repeated short-term reversible ischemia leads to the development of the concept of stress adaptation [2]. After myocardial infarction (MI), left ventricular (LV) enlargement is a
frequent development and this process is known as postinfarction ventricular remodeling. Capillary density becomes
lower in the border zone than in the remote areas of the
infarcted ventricle [3]. The development of coronary collateral circulation in the infarcted myocardium is the heart’s
own bypass mechanism (adaptation) by which it retains the
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blood supply to the myocardium at risk [4]. Thus, it is
extremely important to develop body’s natural angiogenic
process in order to create increased circulation in areas where
blocked coronary arteries deprive the heart muscle of sufficient blood flow, e.g., in the settings of myocardial ischemia
[5].
Ischemia or coronary artery occlusion has been shown to
induce vascular endothelial growth factor (VEGF) mRNA in
rat hearts [6]. VEGF is a well known endothelial cell-specific
angiogenic factor and also a critical regulator of angiogenesis
that stimulates proliferation, migration, and proteolytic activity of endothelial cells [7]. Additional reports suggested
VEGF induced expression of Bcl-2 which eventually functions to enhance the survival of endothelial cells in the toxic,
oxygen-deficient environment [8]. This report points out that
enhanced level of VEGF may have some role in the inhibition
of cardiomyocyte and endothelial cell apoptosis. In addition
to the most popular Bcl-2 family which plays a significant
role in all aspects of cell death and survival, another group of
proteins/molecules (inhibitor of apoptosis, IAP) were also
found to play a prominent role in the inhibition of apoptosis
[9–11]. The most prominent member of this family is survivin, which has been characterized as mammalian IAP. It is
a bifunctional protein and is involved in suppression of apoptosis and cell division. The importance of the expression of
this anti-apoptotic protein survivin, during myocardial adaptation, was never studied so far.
In the present study we showed for the first time significant upregulation of the expression of this anti-death molecule survivin along with the expression of Bcl-2 in the
ischemic preconditioned myocardium subjected to myocardial infarction. Several important transcription factors such
as Pax-5, TFIID, SP1, Stat3, NFjB, were also found to be
activated and involved in IP mediated myocardial angiogenesis. This report also demonstrates for the first time that IP
reduced VEGF-mediated vascular permeability by downregulation of c-Src protein expression and reduction of
edema formation leading to cardioprotection.
Therefore, this study was undertaken to investigate the
possibility of long-term cardioprotective effects of IP in the
post-MI in vivo rat model involving permanent coronary
occlusion. The aim of this study was to demonstrate the
extent to which IP is capable of exerting long-term cardioprotection through preserving cardiac function after MI and
possibly retard the development of overt heart failure. We
also sought to investigate whether augmentation of the myocardial angiogenic response in the form of increased activation of angiogenic (VEGF) and anti-apoptotic proteins
(Bcl-2 and survivin) and transcription factors (Stat3, Pax-5,
NFjB, SP1) could stimulate angiogenesis in the form of
increased capillary and/or arteriolar density and ultimately
contribute towards cardioprotection. Moreover, this study
also determined cardioprotection by c-Src inhibition by IP
compared to control because suppression of Src decreases
vascular permeability that minimizes heart injury.
2. Materials and methods
2.1. Animals
All animals used in this study received human care in
compliance with the principles of laboratory animal care
formulated by the National Society for Medical Research and
Guide for the Care and use of Laboratory Animals published
by NIH.
2.2. Surgical procedure
Male Sprague Dawley rats weighing between 250 and
300g was anesthetized with ketamine HCl (100 mg/kg i.p.)
and xylazine (10 mg/kg i.p.). Cefazolin (25 mg/kg i.p.) was
administered as preoperative antibiotic cover. After tracheotomy and initiation of ventilation (room air, Harvard Apparatus Rodent Ventilator:Model 683), the heart was exposed
through a left lateral thoracotomy (4th intercostal space). A
6-0 polypropylene suture was passed with tapered needle
under the left anterior descending coronary artery (LAD) just
below the tip of the left auricle, and a non-traumatic occluder
was applied on the artery. The myocardium was preconditioned by carrying out a short duration of temporary regional
ischemia (5 min) followed by a period of reperfusion
(10 min) repeated four times (4 × PC). Myocardial infarction
was produced by permanent LAD occlusion. In the non-PC
group, the rats underwent the LAD ligation after opening the
chest for 60 min without the PC procedure. The rats in the
sham group underwent the same procedure except for the
LAD ligation. After completion of all surgical protocols, the
chest wall was re-closed. After application of buprenorphine
(0.1 mg/kg s.c.), and weaning from the respirator, the rats
were placed on a heating pad while recovering from anesthesia.
2.3. Hemodynamics
After surgical procedures (2, 4, 7, 14 and 21 days), the rats
were anesthetized and ventilated as described above. The
arterial catheter was advanced into the left ventricle through
the right carotid artery. Hemodynamics were measured using
PowerLab equipment with Chart v5 software (ADInstruments, Castle Hill, Australia) at a sampling rate of 1000/s,
and averaged over three consecutive cycles. For the assessment of contractile reserve in infarcted myocardium, dobutamine stress test was performed. After resting left ventricular functional study, dobutamine was administered
intravenously at a dose of 5 µg/kg body weight /minute for
3 min.
2.4. Measurement of perfused capillary and arteriolar
density
To measure the perfused capillary density, animals were
perfused just before sacrifice in vivo with FITC conjugated
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
BS1-lectin staining to identify perfused vascular endothelium (Vector Laboratories) 2, 4, 7, 14 and 21 days after
permanent LAD occlusion. For determination of arteriolar
density, cross sections of paraffin-embedded specimens were
stained with FITC conjugated mouse monoclonal anti-a
smooth muscle actin antibody. For the quantitative purpose,
the number of perfused capillary and arteriolar density were
counted on area at risk from the endocardium through the
epicardium of the mid portion of the left ventricular free wall.
Four sections from each heart were examined.
2.5. Measurement of infarct size
2, 4, 7, 14 and 21 days after infarction (n = 5), infarct size
and area at risk were measured as previously described [12].
The infarct size was measured in the 8 horizontal sections
between the point of ligation and the apex. The area at risk
was recognized as the area demarcated with 5% Evans blue
dye, whereas the non-infarcted and infracted areas were
demarcated after incubation with 1% triphenyltetrazolium
chloride (TTC) phosphate buffered solution (pH 7.4) at 37°C
for 15 min and fixed in 10% formalin to increase the contrast
of the Evans blue and TTC staining. With the use of NIH
imaging, the volumes of infarcted myocardium were calculated. Infarct size was reported as a percent of the area at risk.
2.6. Myocardial blood flow
2, 4, 7, 14 and 21 days after permanent LAD occlusion,
myocardial blood flow in the area at risk was measured with
stable gold labeled microspheres (BioPAL, Worcester, MA).
Approximately 1,000,000 microspheres were injected into
the LV. Simultaneously, a reference blood sample was withdrawn from an arterial catheter (0.5 ml/min). At the end of
the experiments the animals were killed by 2 mEq KCl
intravenously. The microscopically visible left ventricular
tissue within 1.5 mm distance from edge of scar tissue were
carefully dissected and weighed. All tissue and blood
samples were sent to Bio PAL and microspheres concentration of each sample were estimate during spectrophotometric
analysis. Myocardial blood flow was calculated as ml/min/g
tissue [13,14].
2.7. Determination of cardiomyocyte and endothelial cell
apoptosis
The formaldehyde-fixed left ventricle (1, 2, 4 and 7 days
after MI) was embedded in paraffin, cut into transverse sections (4 µm thick) and deparaffinized with a graded series of
xylene and ethanol solutions. Immunohistochemical detection of apoptotic cells was carried out using TUNEL in which
residues of digoxigenin-labelled dUTP are catalytically incorporated into the DNA by terminal deoxynucleotidyl transferase, an enzyme which catalyzes a template-independent
addition of nucleotide triphosphate to the 3′-OH ends of
double- or single-stranded DNA [15]. The incorporated
549
nucleotide was incubated with a sheep polyclonal antidigoxigenin antibody followed by a FITC-conjugated rabbit
anti-sheep IgG as a secondary antibody as described by the
manufacturer (Apop Tag Plus, Oncor Inc., Gaithersburg,
MD). The sections (n = 5) were washed in PBS three times,
blocked with normal rabbit serum and incubated with mouse
monoclonal antibody recognizing-a-sarcomeric actin
(Sigma Japan, Tokyo, Japan) followed by staining with
TRIRC-conjugated rabbit anti-mouse IgG (200:1 dilution,
Dako Japan, Tokyo, Japan) [16]. For detection of apoptosis in
endothelial cells the sections were first stained with TUNEL
(FITC staining). The sections were then incubated with rabbit polyclonal anti-von Willebrand factor (Dako) as a primary
antibody followed by incubation with tetrarhodamine
isothiocyanate-conjugated goat anti-rabbit IgG as a secondary antibody. The fluorescence staining was viewed with
confocal laser microscopy (Fluoview, Olympas, Tokyo, Japan). For the quantitative purpose, the number of TUNELpositive cardiomyocytes and endothelial cells were counted
on 100 high power fields (HPF, magnification × 600) from
the endocardium through the epicardium of the mid portion
of the left ventricular free wall in five sections from each
heart [12,17]. Representative confocal images show von
Willebrand factor-positive endothelial cells (strong red staining in their cytosol) which are negative for TUNEL staining
(absence of green staining in the nucleus) as well as those
positive for TUNEL staining (magnification ×1200).
2.8. Protein/DNA array
Nuclear extracts from the left ventricular tissue 2 days
after MI were prepared with the commercially available kit
(Panomics Nuclear Extraction Kit). Each nuclear extract
sample (3–5 µg/µl, 5 µl) were mixed with TranSignal Probe
Mix (10 µl, from Panomics) and dH2O, 5 µl, this mixture was
incubated at 15°C for 30 minutes. 2% agarose gel was prepared in 0.5 × TBE, sample was loaded and were run for 15
min at 120 V. The gel area was excised that contains
protein/DNA complex and were transferred to 1.5 ml tube.
One ml of Extraction buffer (supplied by the company, Panomics, Redwood city, CA) A was added and incubated at
55–60°C. 6 µl of gel extraction beads were added and incubated at room temperature for 10 min. The mixture was
centrifuged at 10,000 rpm for 30 sec to pellet out the beads.
The beads were washed and resuspended the pellet in 150 µl
of extraction buffer B (supplied) and centrifuged at
10,000 rpm for 30 sec. Pellets were dried (air) for 10 min.
Bound probe was eluted by resuspending the pellet in 50 µl of
dH2O with a pipet and incubate at room temperature for
10 min. After centrifugation at 10,000 rpm for 1 min, supernatant was transferred to a fresh 0.2 tube. Eluted probe was
stored on ice until proceeding to hybridization. This labeled
probe was hybridized to the array membrane (Panomics,
Catalog No. MA1010, Redwood City, CA). After hybridization and washing the membrane were treated with Enhanced
Chemi-Luminescence (ECL from Amersham) reagent and
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the transcription factors were detected by autoradiography
for variable lengths of time with Kodak X-Omat film.
2.9. Gel-Shift analysis
To determine SP1, AP-1, NFjB, STAT3, TFIID and Pax-5
binding activity, nuclear proteins were isolated from the heart
of sham, CMI and IPMI groups. Sp1 (5′ GAT-CCT-TCTGGG-AAT-TCC-TAG-ATC 3′), AP-1 (5′ CGC-TTG-ATGAGT-CAG-CCG-GAA-3), NFjB(5′ AGT-TGA-GGG-GACTTT-CCC-AGG-C-3′), STAT3 (5′GAT-CCT-TCT-GGG-AATTCC-TAG-ATC 3′), TFIID (5′GCA-GAG-CAT-ATA-AGGTGA-GGT-AGG-AGG-A 3′) and Pax-5(5′GAA-TGG-GGCACT-GAG-GCG-TGA-CCA-CCG-3′) oligonucleotides were
labeled using T4 polynucleotide kinase as described [18].
2.10. Western blot analysis for VEGF, Bcl-2, Survivin
and c-Src
To quantify the abundance of the angiogenic factor VEGF
and anti-apoptotic factors Bcl-2, survivin and cytoplasmic
tyrosine kinase, c-Src (we included 1day data only for c-Src
study) 2, 4, 7, 14 and 21 days after MI, we performed
standard Western blot using specific antibodies (Santa Cruz
Biotech, Santa Cruz, CA) as previously described [19].
2.11. Src inhibitor
The Src-inhibitor PP1 (BIOMOL, Plymouth Meeting,
Pennsylvania) was used for our experimental purposes. PP1
or vehicle in control was injected (IP) at 1.5 mg/kg [20]
immediately after permanent LAD occlusion.
2.12. Measurement of % water content
Myocardial water content of left ventricle was measured
after 2, 4 and 7 days following MI as previously described
[21]. Left ventricular water content was calculated as percentage difference between initial wet and dry weights after
48 hours incubation at 60°C.
2.13. Statistical analysis
Results are expressed as mean± standard error of the mean
(±SEM). Differences between groups were tested for statistical significance by two-way analysis of variance (ANOVA)
and Student’s t-test (p < 0.05).
3. Result
3.1. Effect of ischemic preconditioning on cardiac function
Pharmacological cardiac stress testing with dobutamine
infusion at 0 and 5 µg/kg/min for 3 min revealed differences
in the extent of cardiac contractile reserve between CMI and
IPMI as shown in Fig. 1A. This was evident from differences
in the extent of change in LVdP/dtmax values displayed by
CMI and IPMI groups during the course of such stress testing
2, 4, 7, 14 and 21 days after surgical intervention. The
differences remained statistically significant after 2, 4, 7,
14 and even after 21 days. The IPMI group displayed significantly elevated contractile reserve at “0”dose point of evaluation compared to the CMI group after 7, 14 and 21 days after
MI. However, this enhanced preservation of contractile reserve in the IPMI group was apparent at 5 µg/kg/min after 2,
4, 7, 14 and 21 days (3128 ± 253 mmHg/s in IPMI group vs.
1808 ± 336mm Hg/s in CMI group at 21 days after MI).
There was no difference in heart rate between the groups
even after 3 weeks of MI (data not shown). Both in CMI and
IPMI left ventricular end-diastolic pressure (LVEDP) was
increased significantly compared to the sham operated
groups, however, in IPMI group the LVEDP at 21 days after
MI was found to be reduced significantly at 5µg/kg/min of
dobutamine (21.2 ± 2.52 vs. 32.4 ± 1.98 mmHg in CMI
group) (data not shown).
3.2. Measurement of infarct size
Heart sections, which were obtained 2, 4, 7, 14 and
21 days after permanent LAD occlusion and stained for the
extent of infarction demonstrated significantly lowered infarct size in the IPMI group (36 ± 2.2% at 2 days post-op)
when compared to the CMI group (53.8 ± 3.7% at 2 days
post-op) (Fig. 1B). This lowered infarction size was well
maintained even after 21 days of post-operation in the IPMI
group.
We found PP1 (1.5 mg/kg/i.p) injection (i.p) just after
LAD ligation in the control MI group can reduce infarct size
significantly when compared to the non-PP1 control group of
hearts (Fig. 9C) following 2 days after MI.
3.3. Effect of ischemic preconditioning on microvascular
Density
3.3.1. (a) Measurement of perfused capillary density
At 400× magnification, 8 non-overlapping random fields,
each selected from non-infarcted, risk area of 4 different left
ventricular sections per sample were examined under fluorescence microscope to determine FITC conjugated BS1lectin labeled capillaries. The IPMI group showed significant
increase in perfused capillary density compared to the control MI group (Fig. 2A). The IPMI group showed significant
difference in perfused capillary density after 2 days post MI.
IPMI group demonstrated increased capillary density (1880
± 210 counts/mm2) compared to the CMI group (1660 ±
296 counts/mm2). The increased level of capillary density in
the IPMI group was also maintained significantly even after
14 days (2650 ± 120 counts/mm2) and 21 days (2760 ± 252
counts/mm2) compared to the CMI group (1940 ±
73 counts/mm2 at 14 days and 2030 ± 185 counts/mm2 at
21 days, respectively).
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
551
A
LVdP/dtmax (mmHg/s)
5000
4000
**
**
**
3000
*
*
2000
*
*
*
1000
0
DOB
(µg/kg/min)
0
5
0
4 days
5
7 days
0
0
5
14days
5
21days
80
%Infarct/Area at risk
B
0
5
2 days
60
40
*
*
*
*
2 days
4 days
7 days
14 days
*
20
0
21 days
Fig. 1. (A) Measurement of LV dP/dt max during dobutamine (5ug/kg/min) stress test for 3min. Changes in LV dP/dt max 2, 4, 7, 14 and 21 days after MI were
recorded with and without dobutamine. Differences were considered significant at p < 0.05. Where * p < 0.05, ** p < 0.01 compared to CMI. [● ] CS, [ C ] IPS,
["] CMI, [◆] IPMI. (B) Infarct size of the hearts, expressed as a percentage of the area at risk 2, 4, 7, 14 and 21 days after LAD occlusion. Results are expressed
as means ± S.E.M of 6 hearts/group. * p < 0.05 compared to CMI. [ ] CMI and [ ] IPMI.
3.3.2. (b) Measurement of arteriolar density
We evaluated the arteriolar density in the non-infarcted
risk area 2, 4, 7, 14 and 21 days after the surgical intervention
(MI). We examined the arteriolar density in the 8 non-overlapping fields in the 4 horizontal sections from middle portion of left ventricle at 400× magnification. The ischemic
preconditioned myocardium showed no change in arteriolar
density even after 4 days after MI however a marked increase
in arteriolar density was observed after 7, 14 and 21 days
after MI when compared to the non-preconditioned myocardium. Arteriolar density remained almost the same in the
CMI group after 7 (2.2 ± 0.8 counts/mm2), 14 (2.2 ±
0.2 counts/mm2) and 21 days (2.3 ± 0.6 counts/mm2) of
infarction. In contrast, in the IPMI group, the arteriolar density increased markedly after 7 (2.7 ± 1.0 counts/mm2), 14
(2.9 ± 0.4 counts/mm2) and 21 days (3.1 ± 0.8 counts/mm2)
compared to the CMI group (Fig. 2B).
3.4. Measurement of blood flow
In the ischemic preconditioned myocardium, significant
increase in regional blood flow was observed with neutron
activation technique just after IP which was maintained even
after 2 days post operation. Four days later the flow in IPMI
group was significantly higher (1.48 ± 0.7 ml/kg/gm) compared to the CMI group (0.89 ± 0.4 ml/kg/gm). This in-
creased flow persisted significantly even after 21 days (1.68 ±
0.2 vs. 1.22 ± 0.7 ml/kg/gm) as shown in Fig. 2C. We also
determined significant increase in blood flow when PP1 (1.5
µg/kg/i.p) was injected to CMI group compared to non-PP1
control following 2 days after MI.
3.5. Extent of apoptotic cell death (cardiomyocyte
and endothelial)
Total number of myocytes in 100 high power fields, which
cover almost 2/3 of mid portion of left ventricular free wall,
was examined for detecting apoptotic cells. In this regional
ischemia model, apoptotic cells are exclusively distributed to
the border area of infarction. Thus, in a border area, apoptotic
cells may exceed 30% at one day after MI, while in other
areas remote from the infarct zone percent apoptotic cells is
virtually zero. Therefore, the data were expressed in
counts/100 high power field and not percent apoptotic cells,
because it is valid only in a global ischemia model. The
number of endothelial and cardiomyocyte apoptotic cells
was significantly high at 1 and 2 days after MI in both groups
(CMI and IPMI), however, in IPMI group the number of
apoptotic cells was significantly lower than that in the CMI
group at each time point. The differences even 4 and 7 days
after MI was significant (Fig. 3A and 3B).
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S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
2800
2600
2400
2200
2000
1800
1600
1400
permanent LAD occlusion
3000
4PC
Perfused Capillary Density
2
( counts/mm )
A
Before
IP
B
**
**
*
2 days
4 days
7 days
4PC
Arteriolar Density
2
( counts/mm )
2.5
2.0
1.5
1.0
0.5
0
C
permanent LAD occlusion
3.5
3.0
Before
IP
*
2 days
4 days
permanent LAD occlusi
on
Blood Flow (ml/min/g)
2.0
1.5
1.0
0.5
**
**
14days
21days
*
*
7 days
14days
**
**
7 days
14days
21days
**
**
*
0
Before
IP
2 days
4 days
21days
Fig. 2. Measurement of Left ventricular perfused capillary (A) and arteriolar density (B) and Regional Blood flow (C) in risk area. 8 non-overlapping random
fields of the tissue sections after in vivo FITC conjugated BS1-lectin perfusion and anti-a smooth muscle actin staining were selected from risk area of four
different left ventricular sections of each hearts (32 fields per heart, 128 fields per group, magnification of 400 × were used, n = 4). Images were captured and
stored in digital tiff file format for image analysis. Counts of capillary density and arteriolar density per mm2 were obtained after superimposing a calibrated
morphometric grid on each digital image using Adobe Photoshop Software. Where * p < 0.05, ** p < 0.01 compared to CMI. ["] CMI and [◆] IPMI. (C)
Regional blood flow in risk area was measured with gold label neutron activation technique. Blood flow was estimated before IP, after IP, 30min, 2 , 4, 7, 14 and
21 days after permanent LAD occlusion. Differences were considered significant at p < 0.05. Where * p < 0.05, ** p < 0.01 compared to CMI. [ ] CMI and
[ ] IPMI.
3.6. Evaluation of protein/DNA array and gel-shift
analysis
Protein/DNA array analysis revealed significant increase
in the DNA binding activity of Sp1 (2.4 fold), NFjB
(1.2 fold), Stat3 (1.7 fold), TFIID (1.65 fold), Pax-5
(1.2 fold) compared to the CMI group. The pro-apoptotic
transcription factor AP-1 was significantly (1 fold) reduced
in the IPMI group (Fig. 4) compared to the corresponding
CMI. This result was again validated by the gel-shift analysis
as shown in Figure 5.
3.7. Expression of VEGF after MI
IP induced VEGF protein expression in IPMI group significantly when compared to the CMI group 7, 14 and
21 days after MI. The protein band density of VEGF in IPMI
group increased with each time compared to the CMI group
even 21 days after operation as shown in Figure 6.
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
553
Fig. 3. TUNEL assay for apoptotic cardiomyocyte (A) and endothelial cells (B). a through d, double immunofluorescence staining for a-sarcomeric actin
(specific for cardiomyocyte; red fluorescence) and TUNEL-positive (green fluorescence). e through h, double immunofluorescence staining for von Willebrand
factor (specific for endothelial cells; red fluorescence) and TUNEL-positive (green fluorescence). a and e, control heart 1 day of post MI; b and f, IPMI heart
1 day of post MI; c and g, control heart 7 days of post MI; d and h, IPMI heart 7 days of post MI. Data was expressed in counts/100 high power field. Bar graph
shows the extent of particular cell death by apoptosis after 1, 2, 4 and 7 days of post MI. Where * p < 0.01 compared to CMI group. [ ] CMI and [ ] IPMI.
3.8. Expression of anti-apoptotic protein Bcl-2
and survivin
The induction of Bcl-2 was observed in both the groups
(CMI and IPMI) as shown in Figure 7 when compared to the
corresponding sham operated group. However, IP upregulated anti-apoptotic protein Bcl-2 in the infarcted myocardium significantly compared to the CMI group and remained
significantly stimulated even 21 days after LAD occlusion.
The figure clearly demonstrated robust expression of Bcl-2 in
the ischemic preconditioned myocardium 2, 4, 7, 14 and
21 days after myocardial infarction when compared to the
baseline, Non-IP sham and IP sham groups. Two days after
MI, in the preconditioned myocardium we found significant
induction of anti-apoptotic protein survivin and the increased
level was maintained up to 7 days after MI as shown in Figure
8 compared to the CMI group. However, after 7 days of post
operation in the preconditioned group we found that the level
of survivin was almost at the baseline (sham) level. These
results suggest earlier anti-apoptotic function of survivin in
the infarcted myocardium. Whereas increased Bcl-2 protein
expression was maintained from day-2 till day-21 in the
IPMI group compared to CMI.
3.9. Expression of c-Src after MI
Western blot analysis revealed significant induction of
c-Src expression (60 kDa) in CMI group right after 1day of
LAD occlusion when compared to sham as well as IPMI
(1day) group. The significant expression of c-Src was maintained in CMI group even after 21 days. However in IPMI
group the expression of c-Src was maintained at baseline or
sham control level throughout the experiment (Fig. 9A).
3.10. Myocardial water content
Src-mediated vascular permeability influences the level of
water content (edema) after MI. We demonstrated that suppression of c-Src by ischemic preconditioning (IPMI) as well
as PP1 decreases the water content of the infarcted myocardium and thus the edema formation significantly as shown in
Figure 9D compared to CMI after 24 hrs of post myocardial
infarction.
4. Discussion
The results of our study documented that ischemic preconditioning can possess angiogenic potential and can improve myocardial blood flow and cardiac function followed
by severe ischemic myocardial injury. Therefore, myocardial
adaptation to intermittent ischemia appears to be a highly
promising approach to induce angiogenesis in a rat model of
myocardial infarction as evidenced by increased capillary
and arteriolar density. This increased micro vascular growth
was found to be associated with a reduced infarct size and
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Fig. 4. Representative Protein/DNA array analysis demonstrates the activation of several transcription factors after two days of post LAD occlusion. Significant
activation of Stat3, Sp1, AP-1, TFIID, Pax-5, NFjB were observed in IPMI group. Where Sham: Control+Sham; CMI: non-preconditioned followed by LAD
occlusion; IPMI: ischemic precondition followed by LAD occlusion.
Fig. 5. Representative gel-shift analysis demonstrates and validates Protein/DNA array result in sham, CMI and IPMI groups. Significant activation of Stat3,
Sp1, TFIID, Pax-5 and NFjB transcription factors were observed in IPMI group compared to CS and CMI groups. However pro-apoptotic transcription factor
AP-1 was found to be significantly reduced in IPMI group compared to CMI group.
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
555
Fig. 6. Representative Western Blots showing the effects of ischemic preconditioning followed by LAD occlusion on the expression of VEGF in rat myocardium
in vivo after sham surgery, 2, 4, 7, 14, 21 days after LAD occlusion. VEGF protein was expressed as 40 kDa. Similar results were obtained in six independent
experiments performed in triplicate. Densitometric scanning from Western blots was expressed as percentage mean value of the baseline control (sham). Where
* p < 0.01 compared to B, † p < 0.01 compared to CMI.
Fig. 7. Representative Western blots showing the effects of ischemic preconditioning followed by LAD occlusion on the expression of Bcl-2 in rat myocardium
in vivo after sham surgery and after 2, 4, 7, 14 and 21 dyas of post MI. Bcl-2 was expressed as 28 kDa. Similar results were obtained in six independent
experiments performed in triplicate. Densitometric scanning from Western blots was expressed as percentage mean value of the baseline control (sham). Where
* p < 0.01 compared to BL, † p < 0.01 compared to CMI.
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S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
Fig. 8. Representative Western blot showing the effect of ischemic preconditioning followed by LAD occlusion on the expression of survivin in rat myocardium
in vivo after sham surgery and after 2, 4, 7, 14 and 21 days of post MI. Survivin was expressed as 16.5 kDa. Similar results were obtained in 4 independent
experiments performed in triplicate. Densitometric scanning from Western blots was expressed as percentage mean value of the baseline control (sham). Where
* p < 0.01 compared to BL, † p < 0.05 compared to CMI.
significant preservation of contractile functional reserve.
Pharmacological cardiac stress testing with dobutamine revealed differences in the extent of cardiac contractile reserve
between IPMI and CMI. The ischemic preconditioned group
displayed significantly elevated contractile reserve compared
to CMI group. LV contractile reserve measured with dobutamine is a sensitive means of detecting differences in the
extent of infarction. Dobutamine stress test is often utilized to
detect the presence of hibernating myocardium in clinical
chronic myocardial ischemia. Angiogenic effects of ischemic
preconditioning in the functional improvements and the decreased infarct size are very general. In hibernating myocardium, the myocytes are viable, but can not contract normally
because of the lack of sufficient blood supply. The presence
of hibernating myocardium indicates that such a myocardial
region is absent from blood supply through the major coronary arteries but is rich in collateral blood vessels as a result
of enhanced angiogenesis. In our experiment, the dobutamine stress test appears to suggest the presence of larger
areas of hibernating myocardium in the ischemic preconditioned heart than in non-preconditioned heart. In the present
study ischemic preconditioning also triggered significant expression of VEGF and increased perfused capillary density
along with increased blood flow. Based on these results, it is
also reasonable to find smaller infarction in the IP heart
because the presence of collateral arteries allows more myocytes to be alive after the cessation of blood supply by LAD
occlusion. However, we can not ascribe all infarct sizelimiting effects of IP to angiogenesis, because direct protec-
tive effects on myocytes of IP can not be eliminated. Such a
direct effect may be observed early after LAD occlusion,
direct effects of IP on myocytes rather than enhanced angiogenesis is a more likely mechanism for early salvage of
myocardium. This may in turn improve contractile function
as assessed by LV dP/dt max. Therefore, infarct size-limiting
effect of IP via direct salvage of myocytes early after LAD
occlusion only negligibly affected contractile performance in
a chronic stage. However, when compared after dobutamine
stress test, IP heart showed greater increase in LV dP/dt max
than non-IP MI hearts, indicative of the presence of larger
number of viable but hibernating myocytes being supplied
blood flow presumably through newly developed collateral
blood vessels in IP heart. The blood flow data also determined increased blood supply in the IPMI group after 2 days
post-op compared to CMI group in this study, which persisted even after 21 days post-op.
To explore the mechanism of this beneficial effect of
intermittent ischemic preconditioning in rat myocardium in
the setting of myocardial infarction we tried to explore the
signal transduction pathway by studying various transcription factors and their involvement with several anti- death
factors in relation to cell death. We know that various transcription factors such as AP-1 [21], SP1 [22] and NFjB [23]
are known to be regulated and found to be important regulator of angiogenesis. Several studies have shown that AP-1
and NFjB are differentially activated by oxygen tension
[24]. Several potential binding sites for the transcription
factors AP-1, AP-2, and SP1 are localized in the VEGF gene
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
557
Fig. 9. (A) Representative Western blot analysis showing the effect of ischemic preconditioning (IP) on the expression of c-Src in rat myocardium in vivo at 1,
2, 4, 7, 14 and 21 day(s) after MI. c-Src was expressed as 60 kDa protein. (B) Effect of Src inhibition on blood flow in control with PP1. Where * p < 0.01
compared to control. (C) Effect of Src inhibition on infarct size in control with PP1. Where * p < 0.01 compared to control. (D) Effect of Src inhibition on infarct
size in control with PP1. Where * p <0.01 compared to control.
[ ] CMI and [ ] CMI+PP1.
promoter and amongst eight glioma cell lines, cellular
mRNA levels of transcription factors SP1 and AP-1 were
found to be closely correlated with those of VEGF [25].
Besides redox regulated transcription factors Stat3, Pax-5
and TFIID were also found to be significantly activated
(except AP-1) by Protein/DNA array analysis (Fig. 4) and
latter validated by gel-shift analysis (Fig. 5) in the IPMI
group after 2 days of LAD occlusion compare to control MI
group. In the ischemic preconditioned group the proapoptotic transcription factor AP-1 was rightly manipulated
and therefore reduced compared to CMI group. Where as
activation of Stat3, in this model demonstrates significant
beneficial effect of ischemic preconditioning mediated cardioprotection in the infarcted heart through the induction of
anti-apoptotic gene Bcl-2 and survivin. However, significant
activation of Pax-5, which is a key regulator of lineagespecific gene expression and TFIID in the setting of MI is yet
to explore.
Now, Angiogenesis induced by the most promising protein, VEGF has been found to be associated with enhanced
cell survival in human umbilical vein endothelial cells in
vitro [26,27]. It is well established that adjunctive local
injection of VEGF demonstrated great promise for many
patients who would not be candidates for any form of revascularization. Currently it is undergoing clinical trial both
as gene therapy and direct administration of protein. In rabbits, myocardial angiogenesis has been demonstrated with
both intra-arterial [28] and intra-muscular [29] injection of
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S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
recombinant VEGF. Controlled formation of new vessels
attenuating or abrogating ischemia or infarction by the administration of VEGF is supposed to be known as therapeutic
angiogenesis. This constitutes a novel treatment option for
the patients who are not suitable for interventions similar as
PTCA or CABG. The temporal pattern of VEGF, Bcl-2 and
survivin expression documented in this study at the protein
level by Western blot analysis correspond to the histological
evidence of angiogenesis in the rat myocardial infarction
model. Our findings suggest that VEGF and Bcl-2 upregulated in response to ischemic preconditioning after 2 days of
myocardial infarction and the expression of both the proteins
stayed high enough significantly at the latter stage of MI
(studied up to 21 days) and functioned endogenously to
promote myocardial angiogenesis.
Recently Kawata et al [30] have also suggested that IP
elevated VEGF gene expression and angiogenesis through
nuclear translocation of PKC e in the infracted myocardium.
These results complement previous studies in which supplemental VEGF and Bcl-2 in vitro and in vivo have been shown
to induce angiogenesis in various tissues and in cell cultures
[17]. Whereas survivin another anti-apoptotic protein downstream of Bcl-2 plays a significant role in the earlier phase
than the latter phase of anti-apoptotic pathways. The expression of survivin was found to be significant after 2 days of MI
in the preconditioned group which increased further compared to the corresponding CMI group and slowly went down
and became normal after 7 days. This result force us to
speculate its role in the earlier phase to protect the myocardium rather than its role in the latter phase of myocardial
infarction.
It is suggested that myocardial infarction leads to persistent post-ischemic vasogenic edema which develops as a
result of increased vascular permeability (VP). This promotes fluid extravasation and interstitial edema, increased
interstitial pressure, collapsing of small vessels and the loss
of perfusion. Hence reducing VP following MI prevent myocardial damage. Vascular leak caused by a variety of myocardial injuries including myocardial ischemia results in myocardial edema, which represents one of major complications
associated with myocardial ischemia. VEGF in response to
ischemic injury promotes vascular permeability (VP). Previous studies demonstrated that Src kinase regulates VEGF
mediated VP in the brain following injury [20]. In our present
study we have determined significant down regulation of
c-Src protein expression in the IPMI heart following MI
compared to CMI. Src kinase activity is known to be increased during the acute phase of preconditioning as a result
of oxidative stress [29]. We presented blocked of Src by PP1
attenuated myocardial edema following MI, leading to reduced infarction and increased blood flow. Therefore, inhibition of c-Src activity in conjunction with VEGF overexpression enhances the benefit of this angiogenic growth factor
while obviating its potential detrimental effect on myocardial
survival and function through an increase in vascular permeability.
In the other hand, VEGF stimulates angiogenesis and play
an important role in prevention of apoptosis of endothelial
cells through upregulation of the most recently described
anti-apoptotic protein, survivin which demonstrated significant expression in the myocardium within 4 days after MI in
the ischemic preconditioned group of animals. This also
documents that this particular protein plays an important role
in the earlier stage due to ischemic preconditioned mediated
trigger of survival pathway. We found that survivin is upregulated within 4 days but it doesn’t stay upregulated for longer
time period. It was almost at baseline level after 1 week of MI
in the preconditioned group of animals. The most important
anti-death candidate Bcl-2 expression also increased within
4 days of MI in the preconditioned group and maintained this
level even after 3 weeks of MI. This observed phenomenon
we think is extremely important for clinical purposes. Survivin perhaps plays a significant role in the initial phase of
angiogenesis by reducing both endothelial and cardiomyocyte cell death. Whereas Bcl-2 expression by ischemic preconditioning might be extremely important throughout MI to
protect from ischemic injury.
Furthermore, we found that capillary as well as arteriolar
densities were also significantly increased in the border zone
of the ischemic myocardium. The capillary density was increased significantly and was maintained even after 3 weeks
of MI compare to CMI. Whereas arteriolar density was found
to be increased after a week of MI and persisted for 21 days.
The extent of myocardial infarction after coronary occlusion
was reduced after 2, 4,7,14 and 21 days after MI significantly
and the amount of viable myocardium was found to be more
abundant in IPMI group compared to the CMI as observed by
cardiomyocyte and endothelial cell survival. Possible
mechanisms to salvage infarcted myocardium in the IPMI
group compared to CMI may be due to increased VEGF
expression which increases coronary flow, opening of latent
collateral vessels by its vasodilating effect, induction of vascular growth and direct cellular protection.
In summary, we observed significant improvements in
regional myocardial function along with increased capillary
and arteriolar density following induction of survival factors
VEGF, Bcl-2 and survivin (first time) in the setting of fully
established chronic rat myocardial infarction model subjected to ischemic preconditioning. Again, transient suppression of Src activity within several days following MI might
reduce ischemia induced heart injury and prevent long term
myocardial damage without disrupting VEGF-mediated revascularization. Thus, co-administration of or sequential
gene therapy with VEGF, Bcl-2 and survivin might prove
beneficial to enhance myocardial collateral blood vessel
function and may represent a new approach to the treatment
of cardiovascular disease.
Acknowledgements
This study was supported by National Institutes of Health
Grant HL 56803 to NM, HL69910 to NM, HL 33889 to DKD
and NATO LST.CLG.979224 to NM and RK.
S. Fukuda et al. / Journal of Molecular and Cellular Cardiology 36 (2004) 547–559
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