Angiogenic Growth Factors in Patients with Cyanotic ...
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Original ArticleKurume Medical Journal, 48,111-116, 2001
Angiogenic Growth Factors in Patients with Cyanotic Congenital Heart Disease and in Normal Children
WAKAKO HIMENO
Department of Pediatrics and Child Health, Kurume University School of Medicine,
Kurume 830-0011, Japan
Summary: Previous studies have demonstrated that the expression of angiogenic growth factors is
induced in hypoxic models. However, little is known about these factors in patients with cyanotic
heart disease. The purpose of this study was to examine the relationship between the plasma level
of angiogenic growth factors and the severity of cyanosis. The study included 85 patients with
cyanotic heart disease and age matched 81 controls. Median age was 4.2 years in the cyanotic
group and 4.8 years in the control group. Mean systemic oxygen saturation was 80.6•}7.3% in the
cyanotic group and 98.1•}0.5% in the control group. In the control group, vascular endothelial
growth factor (VEGF) in the neonatal period was significantly elevated, then rapidly decreased
within 3 months after birth. After 3 months of age, VEGF levels remained at a plateau. In contrast,
this age dependency did not occur in hepatocyte growth factor (HGF) levels. Although VEGF and
HGF levels were not different between the cyanotic and control groups within 3 months after birth,
the VEGF level in the cyanotic group after 3 months of age was significantly elevated compared to
the levels measured in the control group (149.2•}105.6 vs. 66.3•}22.5 pg/ml, p<0.0001). Moreover,
the VEGF level was negatively correlated with oxygen saturation (y=440.6-3.53x, R=0.47, p<
0.0001) in cases more than 3 months old. In contrast, no correlation was found between HGF level
and oxygen saturation. Although physiologically increased VEGF in the neonatal period was rapidly
decreased under normal oxygen saturation, a higher VEGF level persisted if systemic hypoxia was
present. Persistently higher VEGF level may be related to the development of systemic to pul-
monary collateral arteries in patients with cyanotic heart disease.
Key words vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), angio-
genesis, cyanotic congenital heart disease, hypoxia
INTRODUCTION
A ngiogenesis is defined as the phenomenon of new capillary vessel formations from the preexisting vasculature, mainly thin vessels [1-3]. It has been demonstrated that angiogenesis is related to physio-logical phenomenon, such as the formation of circu-latory organs in the fetal period [4], changes in uterine mucous membrane in adult females or tissue repair [5] etc. In these regards, vascular endothelial
growth factor (VEGF) and basic fibroblast growth factor (bFGF) are considered to induce an angiogenic response via their direct effect on endothelial cells
[6,7]. Similarly, recent studies revealed that hepato-cyte growth factor (HGF) strongly affects angiogene-sis [8]. Administration of angiogenic growth factors as in recombinant protein therapy or gene transfer may be augmented in animal models of myocardial and limb ischemia [9].
Recent studies have revealed that VEGF expres-sion is induced by hypoxia [10-15] . Children with cyanotic congenital heart disease often experience the development of aortopulmonary collateral vessels, enlarged bronchial arteries, or major aortopulmonary collateral arteries: (MAPCA) [16-19]. Sandra and colleagues recently reported that children with
Received for publication December 19, 2000Corresponding author: Wakako Himeno, MD, Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-machi, Kurume 830-0011, Japan. Tel: 0942-35-3311(3656) Fax: 0942-38-1792 e-mail: [email protected]
112 HIMENO
cyanotic congenital heart disease have elevated systemic levels of VEGF [20]. We hypothesized that one or both of these angiogenic factors, VEGF and HGF, was related to the abnormal angiogenesis demonstrated by children with cyanotic congenital heart disease. In this study we evaluated plasma levels of VEGF and HGF in patients with cyanotic heart disease and compared them with the plasma levels in healthy subjects to determine whether these angiogenic factors were elevated in the presence of cyanosis.
MATERIALS AND METHODS
This study involved 85 patients with cyanotic
heart disease and age matched 81 control subjects.
The cyanotic group consisted of 39 boys and 46 girls,
and the control group consisted of 43 boys and 38
girls. Median age was 4.2 years (range, 0 days-40
years) in patients with cyanotic heart disease, and 4.8
years (range, 5 days-31 years) in healthy control sub-
jects. Mean systematic oxygen saturation was 80.6•}
7.3% for the cyanotic group, and 98.1•}0.49% in the
control group. Clinical diagnosis in the cyanotic
group are listed in Table 1. The control group con-
sisted of healthy subjects without heart disease
(patients after Kawasaki disease without coronary
abnormality, subjects with an innocent murmur, and
normal neonates).
Blood samples were collected from the peripheral
vein or the inferior vena cava at the time of cardiac
catheterization prior to heparin infusion. In 10 cases
in the cyanotic group, blood samples were collected
from variable sites, such as the inferior vena cava,
peripheral vein, the superior vena cava, a hepatic
TABLE 1.
Clinical diagnosis in cyanotic group
PA: pulmonary atresia; VSD: ventricular septal defect; DORV: double-outlet right ventricle; PA/IVS: pulmonary
atresia/intact ventricular septum
vein, and a systemic artery in order to evaluate the
dispersion of these factors. Blood was collected as
plasma, immediately separated by centrifugation, and
was then kept frozen at -80•Ž until assayed. The
analysis was performed by enzyme immunoassays,
using commercially available kits (human VEGF
Quantikine, R&D Systems, Minneapolis, USA, and a
rat HGF EIA kit, Institute of Immunology, Tokyo,
Japan). The degree of aortopulmonary collateral
arteries development was evaluated by descending
aortography and graded on a 4-point scale according
to Wemovsky et al. [16].
Statistical analysis
All results were expressed as mean value•}
standard deviation. Statistically significant differ-
ences among the sampling sites were evaluated by
ANOVA. A Mann-Whitney test was used for the
comparison between the two groups. Standard linear
or polynominal regression analysis was performed to
identify correlation among the variables. A value of
p<0.05 was interpreted to denote statistical signifi-
cance.
RESULTS
Dispersion of VEGF and HGF levels
Plasma concentration of VEGF and HGF at each
site is shown in Fig. 1. There was no statistical sig-
nificance among the sampling sites.
VEGF and HGF levels in the control group
Plasma concentrations of VEGF and HGF in the
Fig. 1. Dispersion of VEGF and HGF levels.
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ANGIOGENIC GROWTH FACTOR IN CHD 113
Fig. 2. VEGF and HGF levels in the control group.
Fig. 3. Comparison between the cyanotic group and control group.
Fig. 4. Correlation between angiogenic growth factors and oxygen saturation.
Kurume Medical Journal Vol.48, No.2, 2001
114 HIMENO
control group are presented in Fig. 2. The significant
age dependency was observed in the VEGF level.
The increased VEGF levels in the neonatal period
rapidly declined within 3 months after birth. The
VEGF level after 3 months of age did not show such
age dependency. HGF did not exhibit such age
dependency.
Comparison between the cyanotic group and control
group
In subjects over 3 months old from both the
cyanotic and control groups, VEGF and HGF levels
did not change with age. The VEGF level of the
cyanotic group was significantly increased over that
of the control group (149.2•}105.6 pg/mL vs. 66.3•}
22.5 pg/mL, p<0.0001) (Fig. 3). In contrast, the HGF
level in the cyanotic group was not significantly
different compared to that of the control group
(0.28•}0.96 ng/mL vs. 0.56•}1.25 ng/mL, p=0.19). In
analyzing all cases, significant negative correlation
was observed between VEGF levels and systemic
oxygen saturation, while there was no significant
correlation between HGF levels and systemic oxygen
saturation (Fig. 4). There were no statistically signif-
icant relationships between the degree of aortopul-
monary collateral arteries development and plasma
VEGF or HGF levels.
DISCUSSION
Patients with cyanotic congenital heart disease often develop aortopulmonary collateral arteries [16-19]. It seems that the development of these aortopul-monary collateral vessels is associated with the severity of the cyanosis. These aortopulmonary col-lateral vessels may be the major cause of remaining left to light shunts and place a hemodynamic burden on the systemic ventricle in patients having Fontan procedure [19]. Therefore, these vessels are usually closed by transcatheter coil occlusion before or after the operation [17]. The mechanism of development of aortopulmonary collateral vessels is not clear. We hypothesized that persistent systemic hypoxia may induce angiogenesis in patients with cyanotic con-
genital heart disease, and, as a result, these aortopul-monary collateral arteries may develop.
In this study, we evaluated the plasma concen-trations of VEGF and HGF in patients with cyanotic congenital heart disease and healthy control subjects to determine whether the presence of cyanosis influ-enced these angiogenic factors.
In our study, the plasma concentration of VEGF
in normal infants was clearly elevated in the neonatal
period, gradually decreasing to the normal range within a few months after birth. Ariadne and col-leagues reported that the plasma level of VEGF rose significantly in early neonatal life compared with the levels in umbilical cord blood [21]. Increase of VEGF in the early neonatal period may reflect the
physiological development of vessels in the neonatal period [22]. Otherwise, it may reflect transient hypoxia at delivery or sudden hemodynamic changes soon after birth, such as the increase of pulmonary blood flow, etc [13,14,23]. In this study, we demon-strated that elevated plasma concentrations of VEGF in the early neonatal period rapidly decreased to the adult range within a few months after birth. This change may be caused by the stabilization of hemo-dynamics or by the end of advanced VEGF expres-sion in the neonatal period. However, plasma HGF did not show such age dependency.
In our study, VEGF levels in the cyanotic group were significantly higher than that in the acyanotic control group over 3 months after birth. Plasma con-centrations of VEGF may be elevated regardless of the presence of cyanosis in the neonatal period, and immediately decrease to the adult range in the nor-mal acyanotic group. Elevated VEGF levels in the neonatal period in patients with cyanosis may be maintained beyond 3 months. Hypoxia could be
considered a strong stimulus for angiogenesis, and leads to an increase of angiogenic factors under hypoxic conditions in the experimental model
[15,24]. Recent reports demonstrated that increased serum VEGF levels in athletes who were trained in high altitude conditions [12]. The role of VEGF as an
angiogenic stimulator is well established, and there-fore VEGF is a candidate for mediating the abnormal
proliferation of blood vessels (i.e. aortopulmonary collateral arteries) in patients with hypoxic condi-tions.
However, HGF levels did not differ between
cyanotic and acyanotic groups, and also did not show
any correlation with oxygen saturation. These results
suggested that VEGF and HGF were probably induced by different mechanisms. If VEGF expres-
sions are linked with abnormal collateral formations in patients with cyanotic heart disease, gene therapy
using VEGF inhibitors would be possible in the
future.
Several limitations were present in this study.
The sample sites, either the peripheral vein or the
inferior vena cava, were not fixed in either the cya-
notic or control group. However, no significant differ-
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ANGIOGENIC GROWTH FACTOR IN CHD 115
ences between VEGF levels of the peripheral vein
and the IVC were found in our study. Another limita-
tion in this study was the variation of circulatory
dynamics of patients in the cyanotic group. The
cyanotic group consisted of patients with various
congenital heart diseases, such as Tetralogy of Fallot,
decreased pulmonary blood flow, or single ventricle
with increased pulmonary blood flow dependent on
its hemodynamic condition. Pulmonary artery pres-
sure and pulmonary vascular resistance varied by
case. The presence of VEGF may depend not only on
systemic oxygen saturation, but other factors. More
detailed studies should be performed in this area.
Immunohistological examination of aortopulmonary
collateral arteries would clarify causes for the
presence of angiogenic growth factors.
CONCLUSIONS
Although these limitations were present, our
study did clarify that the physiologically increased
VEGF in the neonatal period rapidly decreased under
normal oxygen saturation, and a higher VEGF level
persisted if systemic hypoxia was present. This may be the reason for the development of systemic-to-
pulmonary collateral arteries in patients with cya-notic heart disease.
ACKNOWLEDGMENTS: The author is grateful to Professor Hirohisa Kato, Chairman, Department of Pediatrics and Child Health, Kurume University School of Medicine, for his reviewing of this manuscript. The author would also like to thank Dr. Teiji Akagi and other colleagues, Division of Pediatric Cardiology, for their helpful assistance.
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