Angiogenic Growth Factors in Patients with Cyanotic

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.

Kurume Medical Journal Vol.48, No.2, 2001

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.


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-


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.

REFERENCES

1. Folkman J, and Klagsbrun M. Angiogenic factors.

Science 1987; 235:442-447.

2. Korpelainen El, and Alitalo K. Signaling angiogenesis

and lymphangiogenesis. Curr Opin Cell Biol 1998;

10:159-164.

3. Ferrara N. Vascular endothelial growth factor: molecular

and biological aspect. Curr Top Microbiol Immunol

1999; 237:1-30.

4. Partanen TA, Makinen T, Arola J, Suda T, Weich HA et

al. Endothelial growth factor receptors in human fetal

heart. Circulation 1999;100:583-586.

5. Kinoshita Y, Hassan S, Kawamura M, Matsushima Y,

Okada A et al. Increased hepatocyte growth factor

content in rat stomach during Omeprazone treatment.

Digestion 1998; 59:102-109.

6. Pepper MS, Ferrara N, Orci L, and Montesano R. Potent

synergism between vascular endothelial growth factor

and basic fibroblast growth factor in the induction of

angiogenesis in vitro. Biochem Biophys Res Commun

1992;189:824-831.

7. Breier G, Albrecht U, Sterrer S, and Risau W.

Expression of vascular endothelial growth factor during

embryonic angiogenesis and endothelial cell differen-

tiation. Development 1992;114:521-532.

8. Van Belle E, Witzenbichler B, Chen D, Silver M, Chang L et al. Potentiated angiogenic effect of scatter fac-

tor/hepatocyte growth factor via induction of vascular endothelial growth factor: The case for paracrine amplifi-cation of angiogenesis. Circulation 1998; 97:381-390.

9. Rivard A, Fabre JE, Silver M, Chen D, Murohara T et al.

Age-dependent impairment of angiogenesis. Circulation

1999; 99:111-120.

10. Shweiki D, Itin A, Soffer D, and Keshet E. Vascular

endothelial growth factor induced by hypoxia may

mediate hypoxia-initiated angiogenesis. Nature 1992;

359:843-845.

11. Sakaki T, Yamada K, Otsuki H, Yuguchi T, Kohmura E

et al. Brief exposure to hypoxia induces bFGF mRNA

and protein and protects rat cortical neurons from pro-

longed hypoxic stress. Neuroscience Research 1995;

23:289-296.

12. Asano M, Kaneoka K, Nomura T, Asano K, Sone H et al.

Increase in serum vascular endothelial growth factor

levels during altitude training. Acta Physiol Scand 1998;

162:455-459.

13. Brogi E, Wu T, Namiki A, and Isner JM. Indirect angio-

genic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas

hypoxia upregulates VEGF expression only. Circulation

1994; 90:649-652.

14. Klekamp JG, Jarzecka K, Hoover RL, Summar ML,

Redmond N et al. Vascular smooth muscle cells and

regulated by hypoxia and dexamethasone. Pediatr Res

1997; 42:744-749.

15. Perkett EA, and Klekamp JG. Vascular endothelial

growth factor expression is decreased in rat lung fol-

lowing exposure to 24 or 48 hours of hyperoxia. CHEST

1998;114:525-535.

16. Wernovsky G, Bridges ND, Mandell VS, Castaneda AR,

and Perry SB. Enlarged bronchial arteries after early

repair of transposition of the great arteries. J Am Coll

Cardiol 1993; 21:465-470.

17. McElhinney DBV, Reddy VM, Hanley FL, and Moore P.

Systemic venous collateral channels causing desaturation

after bidirectional cavopulmonary anastomosis:

Evaluation and management. J Am Coll Cardiol 1997;

30:817-824.

18. McFaul RC, Tajik AJ, Mair DD, Danielson GK, and Seward JB. Development of pulmonary arteriovenous

shunt after superior vena cava-right pulmonary artery (Glenn) anastomosis. Circulation 1997; 55:212-216.

19. Triedman JK, Bridges ND, Mayer JE, and Lock JE.

Prevalence and risk factors for aortopulmonary collateral

vessels after Fontan and bidirectional Glenn procedures.

J Am Coll Cardiol 1993; 22:207-215.

20. Starnes SL, Duncan BW, Kneebone JM, Rosenthal GL,

Jones TK et al. Vascular endothelial growth factor and

basic fibroblast growth factor in children with cyanotic


116 HIMENO

congenital heart disease. J Thorac Cardiovasc Surg 2000;

119:534-539.

21. Malamitsi-Puchner A, Tziotis J, Protonotariou E, Xyni K, Sarandakou A et al. Heparin-binding angiogenic factors (basic fibroblast growth factor) in early neonatal life. Pediatr Res 1999; 45:877-880.

22. Lassus P, Ristimaki A, Ylikorkala O, Vinikka L, and

Andersson S. Vascular endotherial growth factor in

human preterm lung. Am Respir Cret Care Med 1999;

159:1429-1433.

23. Millauer B, Wizigmann-Voos S, Schrurch H, Martinez R, Moller NP et al. High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis. Cell 1993; 72:835-846.

24. Tuder RM, Flook BE, and Voelkel NF. Increased gene

expression for VEGF receptors KDR/Flk and Flt in lungs exposed to acute or to chronic hypoxia. J Clin Invest 1995; 95:1798-1807.


Documents

Angiogenic Growth Factors in Patients with Cyanotic