Journal of the American College of Cardiology
© 2000 by the American College of Cardiology
Published by Elsevier Science Inc.
Vol. 35, No. 4, 2000
ISSN 0735-1097/00/$20.00
PII S0735-1097(99)00632-4
Myocardial Infarction
Expression of Vascular
Endothelial Growth Factor in
Patients With Acute Myocardial Infarction
Yukihiro Hojo, MD, PHD, Uichi Ikeda, MD, PHD, Yun Zhu, MD, Motoi Okada, MD,
Shuichi Ueno, MD, Hiroshi Arakawa, MD, Hideyuki Fujikawa, MD, PHD, Taka-aki Katsuki, MD, PHD,
Kazuyuki Shimada, MD, PHD
Tochigi, Japan
OBJECTIVE
The purpose of this study was to investigate the clinical significance of vascular endothelial
growth factor (VEGF) in acute myocardial infarction (AMI). We also examined the
involvement of peripheral blood mononuclear cells (PBMCs), which are a possible source of
VEGF in AMI.
BACKGROUND VEGF is a potent endothelial cell–specific mitogen and could affect the outcome of AMI.
METHODS
Thirty patients with AMI were used for this study. Serum and PBMCs were isolated from
peripheral blood on days 1, 7, 14 and 21 after the onset of AMI. PBMCs were cultured at
a density of 5 ⫻ 106 cells/ml for 24 h. VEGF levels in serum and the culture media were
measured by enzyme-linked immunosorbent assay using a specific anti-human VEGF
antibody.
RESULTS
Serum VEGF levels elevated gradually after the onset of AMI and reached a peak on day 14.
VEGF levels in the culture medium of PBMCs after incubation for 24 h (PBMC-VEGF)
were maximally elevated 7 days after the onset. Maximum serum VEGF levels showed
significant positive correlations with maximum creatine phosphokinase (CPK) levels (r ⫽
⫹0.70, p ⬍ 0.001), but maximum PBMC-VEGF levels did not correlate with maximum
CPK levels. Patients showing improvement in left ventricular systolic function during the
course of AMI showed significantly higher PBMC-VEGF levels than patients without
improvement.
CONCLUSIONS The extent of myocardial damage contributes to the elevation of serum VEGF levels in AMI.
VEGF produced by PBMCs may play an important role in the improvement of left
ventricular function by promoting angiogenesis and reendothelialization after AMI. (J Am
Coll Cardiol 2000;35:968 –73) © 2000 by the American College of Cardiology
Growth factors play important roles in the pathogenesis of
cardiovascular diseases, such as the development of atherosclerosis and restenosis after coronary angioplasty, by regulating cellular proliferation, migration, differentiation and
apoptosis (1,2). Vascular endothelial growth factor (VEGF)
is a potent mitogen for endothelial cells. VEGF has been
reported to promote collateral formation in ischemic cardiac
muscles (3–7) and tissue repair after wounding (8). In
animal models, application of recombinant VEGF to ischemic limbs induced angiogenesis and improved tissue perfusion (9). Recently, Baumgartner et al. (10) demonstrated
From the Department of Cardiology, Jichi Medical School, Minamikawachimachi, Tochigi, Japan.
This study was supported by Research Grants for Cardiovascular Diseases (#10C-1,
#11C-1) from the Ministry of Health and Welfare; a Research Grant (#10670675)
from the Ministry of Education, Science, Sports and Culture; and the Takeda
Medical Foundation.
Manuscript received February 5, 1999; revised manuscript received October 15,
1999, accepted November 22, 1999.
that intramuscular injection of naked plasmid DNA encoding VEGF improved limb ischemia by promoting angiogenesis in patients with critical peripheral arterial disease.
Previously, Seko et al. (11) found that patients with acute
myocardial infarction (AMI) have elevated circulating
VEGF levels compared with normal subjects. However, the
mechanisms and clinical significance of VEGF during AMI
are not fully understood. The course of AMI is a complex,
dynamic process involving not only myocardial cells but also
other cells, such as vascular smooth muscle cells (VSMCs),
endothelial cells and circulating blood cells. Various types of
cells, including cardiocytes and VSMCs, have been shown
to be synthesis sites for VEGF. In addition, blood cells such
as megakaryocytes (12), lymphocytes, macrophages (13) and
neutrophils (14) can secrete VEGF. In the present study, we
investigated the clinical significance of VEGF in patients
with AMI. We measured serum VEGF levels in these
patients and also focused on the role of peripheral blood
Hojo et al.
VEGF in Patients With AMI
JACC Vol. 35, No. 4, 2000
March 15, 2000:968–73
Abbreviations and Acronyms
AMI ⫽ acute myocardial infarction
CAG ⫽ coronary angiography
CPK ⫽ creatine phosphokinase
ELISA ⫽ enzyme-linked immunosorbent assay
LVEF ⫽ left ventricular ejection fraction
LVG ⫽ left ventriculography
PBMCs⫽ peripheral blood mononuclear cells
PTCA ⫽ percutaneous transluminal coronary angioplasty
VEGF ⫽ vascular endothelial growth factor
VSMCs⫽ vascular smooth muscle cells
mononuclear cells (PBMCs), which are one of the most
important sources of VEGF.
METHODS
Patients. We studied 30 AMI patients (25 males and 5
females, aged 61.6 ⫾ 10.7 years, ranging from 40 to 80
years) admitted to Jichi Medical School Hospital from
August 1997 to July 1998. Diagnosis of AMI was based on
the presence of ST segment elevation on the electrocardiogram and a plasma creatine phosphokinase (CPK) level
more than double the normal value. We also studied 12
healthy subjects as controls (nine males and three females,
aged 55.3 ⫾ 4.7 years, ranging from 34 to 72 years). All
patients, with proper indications, received emergent coronary angiography (CAG) and primary percutaneous transluminal coronary angioplasty (PTCA). Left ventriculography (LVG) was performed immediately after primary
PTCA. LVG was not performed in patients with an
increased plasma creatinine level (from 2.0 to 2.5 mg/dL).
Heparin and nitroglycerin were administered intravenously
during PTCA. Administration of heparin was continued for
one to two days after admission. Aspirin was given to all
patients. The use of ␤-blockers, calcium channel blockers,
angiotensin-converting enzyme inhibitors and nitrate preparations was decided by the attending physician. None of
the AMI patients had severe heart failure, hepatic failure,
renal failure or apparent infectious disease. Re-CAG and
LVG were performed before discharge. The left ventricular
ejection fraction (LVEF) was evaluated by LVG using an
area-length method (15).
Blood collection. In our preliminary study, systemic administration of heparin markedly decreased circulating
VEGF levels, but this effect was transient and normal levels
were recovered 24 h after heparin administration. We also
found that VEGF levels in plasma isolated using heparin as
an anticoagulant were underestimated (10.0 ⫾ 2.5%) compared with those in serum or plasma isolated without
heparin. Thus, all blood samples were obtained under
conditions without using heparin.
All patients gave written informed consent. Peripheral
blood was drawn from patients at the time of admission and
969
before administration of heparin (day 1). Peripheral blood
was also obtained 7, 14 and 21 days after the onset of AMI.
At the time of blood sampling, heparin were not administered to any patients. Serum was obtained by centrifugation
and stored at – 80°C until being assayed for VEGF levels.
Attending doctors also obtained blood samples separately at
the time of admission and every 4 h until the maximum
CPK level was determined.
Isolation of PBMCs. Anticoagulated peripheral blood
(20 ml total, containing 0.5 ml heparin) was obtained from
patients with AMI on day 1 (the time of admission ), 7, 14
and 21 days after the onset and also from normal control
subjects. The blood was layered onto Mono-Poly resolving
medium (Dainippon Pharmaceutical Co., Ltd., Osaka,
Japan), and centrifuged at 400g for 20 min at room
temperature. The PBMC fraction was drawn up and rinsed
with phosphate-buffered saline (0.01 mol/liter sodium
phosphate, 0.14 mol/liter NaCl, pH 7.2). After it was
centrifuged again at 400g for 12 min at room temperature,
the cells were resuspended in 1 ml of RPMI medium
containing 10% FBS. The PBMCs were cultured in the
same medium at a density of 5 ⫻ 106 cells/ml in a
humidified atmosphere of 5% CO2 at 37°C. The supernatants were collected 6, 12 and 24 h after incubation and
stored at – 80°C until being assayed.
VEGF assay. VEGF concentrations in serum and the
culture media were determined using a specific enzymelinked immunosorbent assay (ELISA) kit (Amersham, International, Buckinghamshire, UK). Measurements were
performed according to the manufacturer’s instructions.
The standard curve was linear from 15.6 to 1,000 pg/ml of
VEGF.
Statistical analysis. Data are expressed as means ⫾ SEM.
Comparisons of VEGF levels between the patients and
control subjects were analyzed by repeated-measures analysis of variance followed by Scheffe’s test. VEGF levels
between the patients with and without improvement in
LVEF were compared by Student’s unpaired t test. A simple
linear regression line was calculated by the least square
method for assessment of the correlations between two
parameters. Values of p ⬍ 0.05 were considered statistically
significant.
RESULTS
Basic characteristics of patients. Of the 30 patients, 10
had anteroseptal, 18 inferior and 2 lateral AMI. No patients
had any previous myocardial infarction. Fourteen patients
had hypertension (47%), 15 hyperlipidemia (50%), and 6
diabetes mellitus (20%). Twenty-six patients were smokers
(87%), and 10 had a family history of coronary artery disease
(33%). The mean time from the onset of symptoms to
admission was 6.8 ⫾ 1.3 h, with a range of 1 to 24 h.
Emergent CAG was performed on all patients. Of the 30
patients, spontaneous recanalization of the infarction-
970
Hojo et al.
VEGF in Patients With AMI
Figure 1. Changes in serum VEGF levels in patients with AMI.
Serum was isolated from peripheral blood without using heparin.
Patients receiving systemic administration of heparin were excluded. The data on day 1 represent serum VEGF levels at the
time of admission. Serum VEGF levels in AMI patients were
significantly higher than those in control subjects on day 14 after
the onset (163 ⫾ 26.2 vs. 68.7 ⫾ 16.4 pg/ml). Solid circles: mean
serum VEGF levels in AMI patients; open circles: mean serum
VEGF levels in control subjects. Values are expressed as mean ⫾
SEM. *p ⬍ 0.05 compared with control subjects.
related artery was recognized in eight patients. PTCA was
performed on 27 patients. Re-CAG was performed on all
patients and revealed that patency of dilated vessels was
preserved in all patients. The mean of maximum plasma
CPK activity was 3,193⫾ 364 IU/liter, ranging from 371 to
8,642 IU/liter.
Serum VEGF levels. Figure 1 shows changes in serum
VEGF levels in patients with AMI from one to 21 days
after onset. Serum VEGF levels in AMI patients showed a
peak elevation on day 14 (163 ⫾ 26.2 pg/ml), which was
significantly higher than the control subjects (68.7 ⫾
16.4 pg/ml). The day on which serum VEGF levels were
maximum in each patient was distributed between days 7
and 21 after the onset of AMI. We did not find a significant
relationship between serum VEGF levels at the time of
admission and clinical markers (duration of symptoms,
ECG findings, LVEF or degree of collateral at the first
CAG).
VEGF production by PBMCs. Peripheral blood was
drawn from patients on days 1, 7, 14 and 21 after the onset
of AMI for isolation of PBMCs. Figure 2 shows VEGF
levels in the supernatant of cultured PBMCs from the
patients on day 7 after the onset of AMI and those from
control subjects. VEGF levels in the supernatant of cultured
PBMCs from the patients with AMI increased with incubation time, and were significantly higher than those of the
control subjects after incubation for 12 and 24 h. We
defined the VEGF level in the supernatant of cultured
PBMCs after incubation for 24 h as “PBMC-VEGF level”
and used this as a marker of the VEGF production ability of
PBMCs.
JACC Vol. 35, No. 4, 2000
March 15, 2000:968–73
Figure 2. VEGF production by PBMCs isolated from AMI
patients and control subjects. Anticoagulated peripheral blood was
obtained from patients with AMI and normal subjects. PBMCs
were isolated using a Mono-Poly resolving medium and cultured
in an RPMI medium supplemented with 10% FBS at a concentration of 5 ⫻ 106 cells/ml in a humidified atmosphere of 5% CO2
at 37°C. The supernatants were collected 6, 12 and 24 h after
incubation. VEGF levels in the culture medium increased timedependently until 24 h after incubation. Solid circles: mean
VEGF levels in the supernatant of cultured PBMCs in AMI
patients seven days after onset; open circles: mean VEGF levels in
the supernatant of cultured PBMCs in normal subjects. Values are
expressed as mean ⫾ SEM. *p ⬍ 0.05 compared with control
subjects.
Figure 3 shows changes in PBMC-VEGF levels in the
course of AMI. PBMC-VEGF levels in the patients were
significantly higher than those in control subjects (129 ⫾
23.1 pg/ml) from 7 to 14 days after onset of AMI, with a
peak on day 7 after onset of AMI (344 ⫾ 63.5 pg/ml).
Figure 3. VEGF levels in the supernatant of cultured PBMCs
over the course of AMI. VEGF levels in the supernatant of
cultured PBMCs after incubation for 24 h were defined as
“PBMC-VEGF levels.” PBMC-VEGF levels in the patients were
significantly higher than those of control subjects (129 ⫾ 23.1
pg/ml) from 7 to 14 days after the onset of AMI, with a peak on
day 7 (344 ⫾ 63.5 pg/ml). Solid circles: mean PBMC-VEGF
levels in AMI patients; open circles: mean PBMC-VEGF levels
in normal subjects. Values are expressed as mean ⫾ SEM. *p ⬍
0.05 compared with control subjects.
Hojo et al.
VEGF in Patients With AMI
JACC Vol. 35, No. 4, 2000
March 15, 2000:968–73
971
Figure 5. Changes in LVEF during the course of AMI. We
performed left ventriculography (LVG) at the time of admission
followed by re-LVG before discharge on 25 patients. LVEFs were
analyzed by LVG. Mean LVEF levels did not change significantly
in the course of AMI (from 54.1 ⫾ 2.6% to 54.6 ⫾ 1.8%).
Figure 4. Correlations between VEGF levels and CPK levels. A,
A significant positive correlation was found between maximum
serum VEGF levels and maximum CPK levels in the course of
AMI (r ⫽ ⫹0.64, p ⬍ 0.001). Patients receiving heparin for over
seven days were excluded because maximum serum VEGF levels
can be underestimated in such patients. B, No significant correlation was found between maximum PBMC-VEGF levels and
maximum CPK levels.
Correlations between VEGF levels and laboratory data.
We then analyzed the correlation between serum VEGF
levels and clinical parameters in AMI. As shown in Figure
4A, a significant positive correlation was found between
maximum serum VEGF and maximum CPK levels (r ⫽
⫹0.64, p ⬍ 0.001). Similarly, a significant positive correlation was found between maximum CPK-MB and maximum serum VEGF levels (r ⫽ ⫹0.62, p ⬍ 0.001). Significant positive correlations were also observed between
maximum CPK and serum VEGF levels on day 7 (r ⫽
⫹0.54, p ⬍ 0.01) and those on day 14 (r ⫽ ⫹0.58, p ⬍
0.001). On the other hand, no significant correlations were
found between maximum PBMC-VEGF and maximum
CPK levels (Fig. 4B).
Correlations between serum VEGF and PBMC-VEGF
levels. We did not find a significant correlation between
maximum serum VEGF and PBMC-VEGF levels (r ⫽
⫹0.27, p ⫽ 0.15). A weak positive correlation was observed
between serum VEGF and PBMC-VEGF levels on day 7
after the onset (r ⫽ ⫹0.40, p ⬍ 0.05). No significant
correlation was observed between each serum VEGF and
PBMC-VEGF level at any other time point after onset of
AMI (data not shown).
VEGF levels and left ventricular function. We performed re-LVG before discharge on 25 patients. Mean
LVEF levels did not change significantly at the time of
admission and before discharge (from 54.1 ⫾ 2.6% to
54.6 ⫾ 1.8%; Fig. 5). We divided 25 patients into two
groups according to the changes in LVEF between the first
and the second LVG: group A (n ⫽ 9), AMI patients who
did not show decreases in LVEF; group B (n ⫽ 16), AMI
patients who showed decreases in LVEF. There were no
significant differences in maximum serum VEGF levels
between the two groups (Fig. 6A). On the other hand,
maximum PBMC-VEGF levels in group A patients were
significantly higher than those of group B patients (916 ⫾
119 vs. 374 ⫾ 70.1 pg/ml, p ⬍ 0.05; Fig. 6B).
DISCUSSION
The present study revealed significant elevation of serum
VEGF levels and VEGF production by PBMCs in patients
with AMI, compared with normal subjects during the acute
and subacute phases of myocardial infarction. A significant
positive correlation between maximum serum VEGF and
CPK-MB levels suggests that the extent of myocardial
infarction is linked to the elevation of circulating VEGF
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Hojo et al.
VEGF in Patients With AMI
Figure 6. VEGF levels and left ventricular function. We divided
25 patients into two groups according to the changes in LVEF
evaluated by LVG. Group A (n ⫽ 9): AMI patients who did not
show decreases in LVEF during the course of AMI; group B (n ⫽
16): AMI patients who showed decreases in LVEF during the
course of AMI. A, There was no significant difference between
maximum serum VEGF levels between group A and B patients
(238 ⫾ 79.8 vs. 135 ⫾ 32.6 pg/ml). B, Group A patients showed
significantly higher maximum PBMC-VEGF levels than group B
patients (916 ⫾ 119 vs. 374 ⫾ 70.1 pg/ml, p ⬍ 0.05).
levels. Similar to our results, Kawamoto et al. (16) reported
that serum VEGF levels were significantly associated with
peak CPK levels in patients with AMI. Previously, Li et al.
(17) reported a sustained increase in VEGF mRNA expression after AMI in the rat model. They observed a marked
increase in VEGF mRNA expression in microvessels at the
infarction edge or new vessels developed in infarcted myocardium from 24 h to six weeks after infarction. Several
cytokines such as interleukin (IL)-6 and IL-8 have been
reported to be elevated in the acute stage of AMI (18 –28).
These cytokines regulate expression of various growth factors. Dembinska-Kiec et al. (29) reported that IL-1␤
enhances VEGF expression in VSMCs, and Brogi et al.
(30) demonstrated that transforming growth factor-␤1 and
platelet-derived growth factor upregulate VEGF expression
in VSMCs. Maximum elevation of serum VEGF levels
were observed on day 14 after onset. It is speculated that
JACC Vol. 35, No. 4, 2000
March 15, 2000:968–73
cytokine-mediated mechanisms are at least partially responsible for the induction of VEGF production in the subacute
phase of AMI.
We also examined VEGF production by PBMCs isolated
from patients with AMI, because PBMCs are an important
source of VEGF (13). Production of VEGF by PBMCs in
patients with AMI from seven to 14 days after the onset was
significantly higher than that of normal subjects. A weak
positive correlation was observed between PBMC-VEGF
and serum VEGF levels in patients with AMI only on day
7 after the onset, suggesting that VEGF produced by
PBMCs does not strongly contribute to circulating VEGF
levels. We could not find a significant correlation between
PBMC-VEGF levels and clinical parameters, such as maximum CPK levels. The regulatory mechanisms responsible
for enhanced VEGF production by PBMCs after AMI are
still unclear. We should also clarify types of mononuclear
cells that are mainly involved in enhanced VEGF production.
We found that patients with improvement in left ventricular systolic function had higher maximum PBMCVEGF levels than those without improvement. Shinozaki et
al. (31) reported increased expression of VEGF mRNA in
macrophages around the site of infarcted myocardium in an
autopsy case of AMI, which was not found in hearts with
old myocardial infarctions. Pearlman et al. (32) demonstrated that direct infusion of recombinant human VEGF
improved global LVEF and regional wall motion after AMI
in the animal model. We only measured VEGF production
by peripheral PBMCs, but if they represent PBMCs infiltrated in the infarcted myocardium, our data suggest that
local VEGF produced by PBMCs in the heart may play an
important role in the restoration of ventricular systolic
function after AMI. Our results also suggest that PBMCs
are more closely involved in the improvement in left
ventricular systolic function after AMI than circulating
VEGF. We could not find any angiographical development
of major collateral vessels after AMI because the patency of
dilated lesions was preserved in all patients. It is speculated
that enhanced VEGF production by locally infiltrated
PBMCs in the infarcted myocardium promotes proliferation of endothelial cells and development of microvessels,
leading to healing of the infarcted myocardium and repair of
the injured endothelium.
Study limitation. In our study, maximum CPK values did
not always reflect infarction size because reperfusion of the
occluded coronary artery occurred at different time points
after the onset of ischemia. Further studies using parameters
that reflect infarction size more precisely should be considered. In addition, only patients who did not receive immediate heparin therapy were included in the present study,
and because the study population had mean LVEF ⬎ 50%
at the time of admission and before discharge, our study did
not assess serum VEGF and PBMC-VEGF levels in certain
subgroups of AMI patients, such as those with large
Hojo et al.
VEGF in Patients With AMI
JACC Vol. 35, No. 4, 2000
March 15, 2000:968–73
infarction and poor LV function. Further studies will have
to address these subpopulations of AMI patients to assess
the relationship between serum VEGF and PBMC-VEGF
levels and the outcome.
Conclusions. Increased serum VEGF levels and enhanced
production of VEGF by PBMCs were found in patients
with AMI. Elevation of circulating VEGF levels may have
a cardiovascular protective effect by promoting angiogenesis
and proliferation of endothelial cells. PBMCs do not
strongly contribute to the elevation of circulating VEGF
levels, but may have an important role in the improvement
of ventricular function by enhancing local VEGF production in the heart during the subacute phase of AMI.
Reprint requests and correspondence: Dr. Uichi Ikeda, Department of Cardiology, Jichi Medical School, Minamikawachimachi, Tochigi 329-0498, Japan. E-mail:[email protected].
Acknowledgment
We thank Toshiko Kambe for her excellent technical
assistance.
13.
14.
15.
16.
17.
18.
19.
20.
21.
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