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Ann Thorac Surg 2003;75:1523-1526
© 2003 The Society of Thoracic Surgeons

Original article: cardiovascular

Vascular endothelial growth factor in children with congenital heart disease

Yoshio Ootaki, MD, PhDa*, Masahiro Yamaguchi, MD, PhDa, Naoki Yoshimura, MD, PhDa, Shigeteru Oka, MD, PhDa, Masahiro Yoshida, MDa, Tomomi Hasegawa, MDa

a Department of Cardiothoracic Surgery, Kobe Children’s Hospital, Kobe, Hyogo, Japan

Accepted for publication November 11, 2002.

* Address reprint requests to Dr Ootaki, 1-1-1 Takakuradai, Suma-ku, Kobe, Hyogo 654-0081, Japan.
e-mail: y.ootaki{at}nifty.ne.jp


   Abstract
Top
 Abstract
Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
 References
 
BACKGROUND: Children with cyanotic congenital heart disease may experience the development of abnormal vessels that become a source of significant morbidity. Abnormal vessel proliferation in these children may take several forms, including systemic-to-pulmonary collateral arteries, systemic-to-pulmonary venous collaterals, systemic venous collateral channels after bidirectional cavopulmonary anastomosis, and pulmonary arteriovenous malformations. However, no entity responsible for these abnormalities has been identified yet. This study determined whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor (VEGF) and whether elevated VEGF correlated with these abnormal vessels.

METHODS: Mean systemic room air oxygen saturation (SpO2), blood cell counts (RBC), and serum VEGF levels were measured preoperatively. Samples were obtained from 61 children with acyanotic heart disease (group N) and 102 children with cyanotic heart disease (group C) before cardiac surgery. Postoperative catheterization was performed 1-month after the operation to evaluate the abnormal vessels in group C.

RESULTS: The VEGF level was significantly elevated in group C (355.0 ± 287.1 pg/mL) compared with group N (203.0 ± 221.6 pg/mL; p < 0.001). VEGF levels in patients with a single ventricle associated with asplenia syndrome (n = 7) in group C were significantly elevated (711.9 ± 443.5 pg/mL) compared with other patients. There was no significant correlation between VEGF level and SpO2 or RBC. Abnormal vessels were diagnosed in 19.6% (20/102) patients in group C. There was no difference in VEGF levels between the patients with abnormal vessels (336.8 ± 182.5 pg/mL) and the patients without abnormal vessels (359.1 ± 306.8 pg/mL).

CONCLUSIONS: Children with cyanotic heart disease have elevated systemic levels of VEGF, especially in those patients with a single ventricle associated with asplenia syndrome. There was no significant relationship in VEGF levels between the patients with abnormal vessels and without these vessels.


   Introduction
Top
 Abstract
 Introduction
Patients and methods
 Results
 Comment
 Acknowledgments
 References
 
Children with cyanotic congenital heart disease may experience the development of abnormal vascular channels that become a source of significant morbidity. Abnormal vessel proliferation in these children may take several forms, including systemic-to-pulmonary collateral arteries [1–4], systemic-to-pulmonary venous collaterals [5], systemic venous collateral channels after bidirectional cavopulmonary anastomosis [6, 7], and pulmonary arteriovenous malformations [8]. However, no entity responsible for these abnormalities has been identified yet. The purposes of this study were to determine [1] whether the children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor (VEGF) [2], whether the VEGF levels correlate with the degree of cyanosis [3], whether the VEGF levels correlate with these abnormal vascular channels, and [4] whether the VEGF levels differ in some diseases.


   Patients and methods
Top
 Abstract
 Introduction
 Patients and methods
Results
 Comment
 Acknowledgments
 References
 
From June 1999 to May 2000, we prospectively studied 163 nonconsecutive children 3-months-old to 16-years-old who were undergoing open-heart surgery for the correction of congenital heart disease. The mean age was 6.0 ± 3.4 years. Patients were excluded if the operation was an emergent case. To increase the number of cyanotic patients, in the latter portion of the study we only measured the VEGF levels in the cyanotic patients. However, the noncyanotic, scheduled patients were consecutive. Informed consent was obtained from each participating patient. Mean systemic room air oxygen saturation (SpO2) was measured preoperatively. Before surgery, blood was analyzed for red blood cell counts (RBC), and serum was analyzed for the VEGF level.

The children were divided into two groups: acyanotic group (group N, n = 61), and cyanotic group (group C, n = 102). The children of group C were divided into four subgroups: functional single ventricle associated with asplenia syndrome (group C-A, n = 7); functional single ventricle except asplenia syndrome (group C-SV, n = 11); pulmonary atresia associated with ventricular septal defect (group C-PA, n = 18); and other cyanotic heart disease (group C-O, n = 66).

Preoperative catheterization was performed for all patients. Postoperative catheterization was also performed 1-month after the operation for all cyanotic patients to detect abnormal vessel proliferation. The abnormal vessels were defined by diameter (> 2.0 mm) and amount of blood flow. When the direct opacification of a pulmonary artery segment with contrast injection into the collateral artery was visible, we occluded it using the coil embolization technique. The systemic-to-pulmonary venous collaterals or systemic venous collateral channels were also defined and occluded with catheter intervention. If there was a risk of migration of the coil, we decided to ligate it under direct visualization.

Blood was collected from the systemic artery just after the induction of anesthesia but before the surgery. Samples were allowed to clot for 30 minutes and were centrifuged at 1000 g for 10 minutes at 4°C. The serum was removed and stored at -70°C. The VEGF levels were measured with the Quantikine VEGF enzyme-linked immunoabsorbent assay kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions.

Statistical analyses were performed with a statistical analysis program (Statview 5.0; Cricket Software, Philadelphia, PA). All values were expressed as mean ± standard deviation. An unpaired Student’s t-test and a one-way repeated-measures analysis of variance were used to assess the differences in VEGF levels. When the differences were determined by the one-way repeated-measures analysis of variance to be significant, the differences were further analyzed by the Scheffé test. The correlation between differences in SpO2, RBC, and VEGF levels were calculated by linear regression analysis. Differences were considered significant at p less than 0.05.


   Results
Top
 Abstract
 Introduction
 Patients and methods
 Results
Comment
 Acknowledgments
 References
 
Mean systemic room air oxygen saturation was 98.7% ± 1.8% in group N and 80.4% ± 9.5% in group C (p < 0.001). Mean red blood cell counts was 477.0 ± 42.7x104/mm3 in group N and 569.7 ± 70.4x104/mm3 in group C (p < 0.001). The VEGF levels in serum were detected in all children. The mean VEGF level was 203.0 ± 221.6 pg/mL in group N and 355.0 ± 287.1 pg/mL in group C (p < 0.001; Fig 1).



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  		Fig 1. The vascular endothelial growth factor (VEGF) levels in the acyanotic group (group N) and cyanotic group (group C).

 
The linear regression analysis revealed a correlation between SpO2 and RBC (r = 0.716, p < 0.001; Fig 2). The coefficient of correlation between SpO2 and the VEGF levels was low (r = 0.283, p < 0.001; Fig 3). The linear regression analysis did not demonstrate any significant correlation between RBC and the VEGF levels (r = 0.142, p = 0.07; Fig 4).



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  		Fig 2. Relationship between systemic room air oxygen saturation (SpO2) and red blood cell counts (RBC).

 


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  		Fig 3. Relationship between systemic room air oxygen saturation (SpO2) and vascular endothelial growth factor (VEGF) levels.

 


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  		Fig 4. Relationship between red blood cell counts (RBC) and vascular endothelial growth factor (VEGF) levels.

 
Postoperative catheterization was performed for all cyanotic patients. Twenty of 102 (19.6%) patients in group C had significant abnormal vessel proliferations that required treatment with catheter intervention or surgery. Fifteen patients had significant systemic-to-pulmonary collateral arteries and coil embolization was performed. Four patients had systemic-to-pulmonary venous collaterals. One patient had systemic venous collateral channels after bidirectional cavopulmonary anastomosis. No patients had pulmonary arteriovenous malformations in this study. The mean VEGF level was 360.8 ± 308.3 pg/mL in the patients without abnormal vessel in group C, and 331.0 ± 179.6 pg/mL in the patients with abnormal vessel in group C (p = 0.68). There was no significant change in VEGF levels between these three types of abnormal vessels.

The VEGF levels in group C-A, C-SV, C-PA, and C-O were 711.9 ± 443.5 pg/mL, 364.1 ± 240.8 pg/mL, 286.9 ± 199.1 pg/mL, and 334.2 ± 274.2 pg/mL, respectively. The VEGF levels in group C-A were higher than C-PA (p < 0.01), C-O (p = 0.01), and C-SV groups (p = 0.08; Fig 5).



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  		Fig 5. The vascular endothelial growth factor (VEGF) levels in subgroups of group C. The VEGF levels in group C-A were higher than C-PA (p < 0.01), and C-O (p = 0.01). There was no significance between the C-A and C-SV groups (p = 0.08). (C-A = functional single ventricle associated with asplenia syndrome; C-O = other cyanotic heart disease; C-PA = pulmonary atresia associated with ventricular septal defect; C-SV = functional single ventricle except asplenia syndrome.)

 

   Comment
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
Acknowledgments
 References
 
VEGF is a potent mitogen acting specifically on vascular endothelial cells, and is known to play a role in angiogenesis in widely divergent circumstances, such as embryonic development [9], wound healing [10], tumor growth [11], rheumatoid arthritis [12], and ischemic retinopathy [13]. VEGF has been demonstrated to induce angiogenesis, endothelial cell proliferation, and migration, thereby promoting blood vessel growth. Recent studies have demonstrated that angiogenesis, facilitated by administration of angiogenic growth factors as in recombinant protein therapy or gene transfer, may be augmented in animal models of myocardial ischemia [14]. Therapeutic angiogenesis with VEGF was recently performed in order to reduce the unfavorable tissue effects caused by ischemia [15]. However, it was still unknown whether VEGF plays an important role in angiogenesis or abnormal vessel proliferation of the congenital heart disease in children.

The present study demonstrated that VEGF level was significantly elevated in children with cyanotic heart disease like a previous study [16]. Hypoxia is a strong stimulus for angiogenesis and leads to an upregulation of VEGF. However, the VEGF level had no significant correlation with the degree of cyanosis in our study. Although hypoxia can be one factor of elevated levels of VEGF, other factors such as cytokines or the function of time might also result in VEGF elevation. VEGF is classified into subgroups and some types of VEGF are not affected by hypoxia [17]. This fact might also be related to the poor relationship seen between the VEGF level and the degree of cyanosis.

The present study also demonstrated that VEGF level had no correlation with the existence of abnormal vessel proliferation. Angiogenesis normally occurs at variable rates in different organs, because it depends on the tissue regeneration. This response is amplified under conditions that result in cellular hypoxia. Although VEGF might promote angiogenesis in children with cyanosis, VEGF had no relation with such visible, large vessels in the catheterization. According to the report of Rivard and associates [18], the blood perfusion of the hindlimb begins to increase at 7 to 14 days after the acute limb ischemia. We speculated that a 1-month duration was enough time to estimate the proliferation of the collateral flow. However, the duration might not be sufficient to estimate the visible, large vessels. Even without major abnormal vessels, patients with cyanotic congenital heart disease frequently have high amounts of collateral flow by numerous small collaterals. These secondarily developed systemic pulmonary collaterals might relate to VEGF levels.

The present study demonstrated that the patients with asplenia syndrome had increased serum VEGF levels. It is not known whether the abnormal vessels are present from birth and become hemodynamically significant after the surgery, or whether they develop de novo in response to the changed pattern of circulation and pressures created by surgery. However, the elevated level of VEGF might stimulate a proliferation of abnormal vessels, which was not visible in the catheterization. Therefore, elevated VEGF levels might be a risk factor after surgery for congenital heart disease especially in patients with asplenia syndrome.

This study was limited by the small number of patients of congenital heart disease with asplenia syndrome. A large number of patients associated with asplenia syndrome might indicate a relationship between VEGF level and the abnormal vessels. A second study limitation results from the fact that the normal range of VEGF levels as a patient ages is uncertain. Rivard and associates [18] reported that angiogenesis responsible for collateral development in limb ischemia is impaired with aging. In that study, the expression of VEGF was impaired in old animals compared with younger animals. Because the range of age in our patients was 3-months-old to 16-years-old, this aging effect may factor into the VEGF level. A third study limitation was the lack of serial VEGF levels before surgery, after surgery, and late-term. To clarify the effects of VEGF on the vascular creation, further investigations will be necessary.

In summary, children with congenital heart disease have elevated systemic levels of VEGF, especially in the patients with single ventricle associated with asplenia syndrome. There was no significant relationship in VEGF levels between the patients with abnormal vessels and without these vessels.


   Acknowledgments
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
References
 
We thank Michael Kopcak for editorial advice.


   References
Top
 Abstract
 Introduction
 Patients and methods
 Results
 Comment
 Acknowledgments
 References
 

  1. Ichikawa H., Yagihara T., Kishimoto H., et al. Extent of aortopulmonary collateral blood flow as a risk factor for Fontan operations. Ann Thorac Surg 1995;59:433-437.[Abstract/Free Full Text]
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  3. McElhinney D.B., Reddy V.M., Tworetzky W., Petrossian E., Hanley F.L., Moore P. Incidence and implications of systemic to pulmonary collaterals after bi-directional cavopulmonary anastomosis. Ann Thorac Suurg 2000;69:1222-1228.
  4. Spicer R.L., Uzark K.C., Moore J.W., Mainwaring R.D., Lamberti J.J. Aortopulmoanry collateral vessels and prolonged pleural effusions after modified Fontan procedures. Am Heart J 1996;131:1164-1168.[Medline]
  5. Clapp S., Morrow W.R. Development of superior vena cava to pulmonary vein fistulae following modified Fontan operation: case report of a rare anomaly and embolization therapy. Pediatr Cardiol 1998;19:363-365.[Medline]
  6. Gatzoulis M.A., Shinebourne E.A., Redington A.N., Rigby M.L., Ho S.Y., Shore D.F. Increasing cyanosis early after cavopulmonary connection caused by abnormal systemic venous channels. Br Heart J 1995;73:182-186.[Abstract/Free Full Text]
  7. McElhinney D.B., Reddy V.M., Hanley F.L., Moore P. Systemic venous collateral channels causing desaturation after bi-directional cavopulmonary anastomosis: evaluation and management. J Am Coll Cardiol 1997;30:817-824.[Abstract]
  8. Srivastava D., Preminger T., Lock J.E., et al. Hepatic venous blood and the development of pulmonary arteriovenous malformations in congenital heart disease. Circulation 1995;92:1217-1222.[Abstract/Free Full Text]
  9. Feucht M., Christ B., Wilting J. VEGF induces cardiovascular malformation and embryonic lethality. Am J Pathol 1997;151:1407-1416.[Abstract]
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