Cardiology in the Young (2010), 20, 505508

r Cambridge University Press, 2010

doi:10.1017/S1047951110000545

Original Article Vascular endothelial growth factor and its soluble receptor in infants with congenital cardiac disease
Anja Pohl-Schickinger,1 Petra Koehne,1 Thomas Schmitz,1 Katharina R. L. Schmitt,2 Michael Hu bler,3 4 5 2,6 Matthias Redlin, Felix Berger, Brigitte Stiller
1

Department of Neonatology, Charite University Medicine Berlin; 2Department of Congenital Heart Disease; Department of Cardiothoracic and Vascular Surgery; 4Department of Anesthesia, Deutsches Herzzentrum Berlin; 5 Department of Congenital Heart Disease, Deutsches Herzzentrum Berlin and Charite University Medicine Berlin, Campus Virchow Klinikum, Berlin, Germany; 6Department of Congenital Heart Disease, Childrens University Hospital, Freiburg, Germany
3

Abstract Patients with cyanotic congenital cardiac disease often develop major aortopulmonary collaterals. Vascular endothelial growth factor is a key promoter of angiogenesis. Its soluble receptor-1 acts as a potent antagonist. We studied 30 infants with cyanotic congenital cardiac disease and 27 infants with acyanotic congenital cardiac disease. Central venous plasma vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 levels were measured before, and 24 and 96 hours after surgery. There was no difference between plasma vascular endothelial growth factor levels in infants with cyanotic and those with acyanotic congenital cardiac disease. In cyanotic infants, the soluble vascular endothelial growth factor receptor-1 levels tended to be higher than in the acyanotic infants. In conclusion, there is no significant difference in the plasma levels of vascular endothelial growth factor and its soluble receptor-1 between infants with cyanotic and those with acyanotic congenital cardiac disease.
Keywords: Angiogenesis; cardiopulmonary bypass; major aortopulmonary collaterals Received: 29 September 2009; Accepted: 14 March 2010; First published online: 17 May 2010

disease often develop major aortopulmonary collaterals. This is a cause of significant morbidity in some children. The molecular mechanisms that trigger major aortopulmonary collateral growth are not yet known. In patients with cyanotic congenital cardiac disease, increased serum vascular endothelial growth factor levels have been reported that correlate with the degree of cyanosis.1 Suda et al2 noted that elevated serum vascular endothelial growth factor returned to normal after biventricular repair in patients with cyanotic congenital cardiac
Correspondence to: Dr A. Pohl-Schickinger, Department of Neonatology, University Medicine Berlin, Campus Virchow Klinikum, AugustenCharite burger Platz 1, 13353 Berlin, Germany. Tel: 0049 30 450 566 122; Fax: 0049 30 450 566 922; E-mail: anja.pohl@charite.de

HILDREN WITH CYANOTIC CONGENITAL CARDIAC

disease. Vascular endothelial growth factor is a specific mitogen for endothelial cells and a key promoter of angiogenesis. It interacts with two endothelial tyrosine kinase receptors.3 The soluble vascular endothelial growth factor receptor-1 acts as a high-affinity antagonist, blocking vascular endothelial growth factor-induced cell proliferation and migration.4 The expression of vascular endothelial growth factor is stimulated by hypoxiainducible factors, transcriptional regulators that mediate cellular response to low oxygen levels. At the same time, hypoxia may induce the abnormal expression of soluble vascular endothelial growth factor receptor-1, which in turn downregulates angiogenesis.5 The role of soluble vascular endothelial growth factor receptor-1 in congenital cardiac disease has not been investigated yet.

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This study aims to compare the plasma vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 levels of infants with cyanotic and acyanotic congenital cardiac disease.

Patients and methods

We studied 30 infants with cyanotic congenital cardiac disease mean systemic oxygenation under room air is less than 92% and 27 with acyanotic congenital cardiac disease mean systemic oxygenation under room air is 95% or higher who underwent cardiopulmonary bypass from October, 2005 to June, 2007 in a single centre. The data collected included demographics, anatomic diagnosis, and details of surgery (Tables 1 and 2). Both groups differed only in median bypass duration, which was significantly longer in the cyanotic group. We used a central venous catheter to obtain haematocrit and blood samples for vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1. Blood samples for vascular endothelial growth factor and soluble
Table 1. Diagnoses and surgical procedures.

vascular endothelial growth factor receptor-1 were collected pre-operatively, and 24 and 96 hours post-operatively in disodium-ethylenediaminetetraacetate and immediately centrifuged for 10 minutes at 3000 g. The plasma was removed and stored at 2808C until the assays were performed. Vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 were measured using commercially available Enzyme-linked immunosorbent assay kits (human VEGF Quantikine and human sVEGF R1/Flt-1 Quantikine, R&D Systems, Minneapolis, Minnesota, United States of America) according to the manufacturers protocol. Statistical analysis was performed using SPSS 12.0 (SPSS, Chicago, Illinois, United States of America). Descriptive statistics are reported as medians with interquartile ranges. We used MannWhitney U and Wilcoxon tests to compare age, weight, oxygen saturation, haematocrit, and levels of vascular endothelial growth factor or soluble vascular endothelial growth factor receptor-1. Differences were considered significant at p-value below 0.05.

Cyanotic (N 5 30) Tetralogy of Fallot/double outlet right ventricle Complete atrioventricular septal defect Ventricular septal defect with or without atrial septal defect Atrial septal defect Tricuspid atresia D-transposition of great arteries, ventricular septal defect, Glenn anastomosis/BlalockTaussig shunt Pulmonary stenosis/pulmonary valve regurgitation Total anomalous pulmonary venous return and atrial septal defect Double inlet left ventricle, ventricular septal defect and pulmonary stenosis Partial anomalous pulmonary venous return Partial anomalous pulmonary venous return, pulmonary stenosis, sinus venosus defect and functional common atrium Tricuspid valve regurgitation and mitral valve regurgitation Left ventricular outflow tract obstruction Right ventricular outflow tract obstruction 16 4 1 4 2 1 1 1

Acyanotic (N 5 27) 2 3 5 4 4 4 2 2 1

Surgical procedures Correction Correction Closure Closure Glenn/shunt/total cavopulmonary connection Fontan Correction Correction, closure Fontan Correction Correction Valve reconstruction Resection Resection

Table 2. Description of the study groups (infants with cyanotic and aycyanotic congenital cardiac disease). Cyanotic (N 5 30) Age (years) Body weight (kg) Body height (cm) Sex (male/female) Pre-operative mean systemic oxygenation under room air Pre-operative haematocrit (%) Median bypass duration (min)
Values are shown as median (interquartile range) except for sex

Acyanotic (N 5 27) 1.3 (0.53.9) 9.0 (5.713.2) 76 (64 99) 16/14 0.99 (0.981.00) 0.37 (0.320.41) 59 (4584)

p 0.104 0.350 0.206 0.429 ,0.001 0.005 ,0.001

0.7 (0.42.6 ) 7.3 (5.011.3) 69 (6289) 11/16 0.85 (0.800.90) 0.43 (0.350.48) 111 (82135)

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VEGF [pg/ml] 200 C-CCD 150 A-CCD

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The Institutional Ethics Committee at Charite University Medicine Berlin approved the study, and all participating parents granted informed consent.

Results Mean systemic oxygenation under room air and haematocrit Table 2 depicts the pre-operative mean systemic oxygenation under room air and haematocrit in the study groups. Haematocrit was significantly higher in the cyanotic group than in the acyanotic group. Vascular endothelial growth factor and soluble vascular endothelial growth factor receptor-1 in the cyanotic and acyanotic groups Pre- and post-operatively, central venous vascular endothelial growth factor did not differ between the cyanotic and acyanotic groups (Fig 1). Vascular endothelial growth factor levels, measured 24 hours post-operatively, were comparable to pre-operative levels in both groups 35.6 (17.545.5) picograms per millilitre in the cyanotic group and 42.4 (10.859.2) picograms per millilitre in the acyanotic group. There were no significant changes in vascular endothelial growth factor levels measured 96 hours post-operatively 42.8 (19.783.5) picograms per millilitre in the cyanotic group and 65.2 (29.7183.5) picograms per millilitre in the acyanotic group. Pre-operatively measured levels of soluble vascular endothelial growth factor receptor-1 tended to be higher in the cyanotic group, 1085.4 (418.62920.6) picograms per millilitre, than in the acyanotic group, 552.6 (207.91798.5) picograms per millilitre (Fig 2). In the cyanotic group, soluble vascular endothelial growth factor receptor1 was comparable to pre-operative levels 24 hours after cardiac surgery, 940.2 (652.02163.5) picograms per millilitre, and remained unchanged until 96 hours post-operatively, 1002.9 (493.32423.3) picograms per millilitre. In the acyanotic group, soluble vascular endothelial growth factor receptor1 levels were elevated 24 hours post-operatively compared to the pre-operative levels, 1125.2 (357.42122.0) picograms per millilitre and returned to pre-operative levels within 96 hours after surgery, 470.0 (222.42025.4) picograms per millilitre. Discussion
In this study, we examined plasma vascular endothelial growth factor and its soluble receptor in infants with cyanotic and acyanotic congenital cardiac disease who underwent open cardiac surgery.

100

50

preoperatively

24h

96h

Figure 1. Central venous vascular endothelial growth factor (VEGF) (picogram per millilitre) measured in patients with cyanotic (C-CCD) and acyanotic congenital cardiac disease (A-CCD) pre-operatively, and 24 and 96 hours after surgery. Values are shown as the median and 75th percentile.
s-Flt1 (pg/ml) 5000 C-CCD 4000 A-CCD

3000

2000

1000

preoperatively

24h

96h

Figure 2. Central venous soluble vascular endothelial growth factor receptor-1 (s-Flt-1) (picogram per millilitre) measured in patients with cyanotic (C-CCD) and acyanotic congenital cardiac disease (A-CCD) pre-operatively, and 24 and 96 hours after surgery. Values are shown as the median and 75th percentile.

In contrast to previous studies,1,2,6,7 we did not find any differences in vascular endothelial growth factor levels between patients with cyanotic and acyanotic congenital cardiac disease and also observed lower vascular endothelial growth factor levels overall. This may be due in part to our different methodical approach. Plasma is the preferred medium for

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measuring free-circulating vascular endothelial growth factor levels, since the highly variable platelet-mediated secretion of vascular endothelial growth factor during the clotting process influences the vascular endothelial growth factor levels.810 However, most previous data refer to vascular endothelial growth factor serum values,1,2,6 which might be influenced by platelet degranulation, thus leading to higher vascular endothelial growth factor levels.10 For our study, we followed a recommended protocol9 using plasma samples that were cooled and immediately centrifuged after collection. In order to optimise comparability, we collected blood samples exclusively from central venous lines. Our patients were also younger than those in previous studies. We found slightly, but not significantly, higher pre-operative plasma levels of soluble vascular endothelial growth factor receptor-1 in infants with cyanotic congenital cardiac disease. This phenomenon is probably explained by the known induction of soluble vascular endothelial growth factor receptor-1 expression under hypoxic conditions, which blocks the physiological vascular endothelial growth factor.5 Soluble vascular endothelial growth factor receptor-1 reduces the basal serum angiogenic activity of vascular endothelial growth factor in vitro,11 since it acts as a negative regulator by sequestering the vascular endothelial growth factor via its ligand-binding domain. Bound vascular endothelial growth factor is not detected by the Enzyme-linked immunosorbent assay. It might therefore be possible that hypoxia-induced vascular endothelial growth factor expression is counterbalanced by higher soluble vascular endothelial growth factor receptor-1 levels.

with congenital cardiac disease before, tended to be higher in cyanotic infants.

Acknowledgements
We thank Sarah Smithson-Compton for suggestions regarding English language usage and Evelyn Strauss for technical assistance.

References
1. Ootaki Y, Yamaguchi M, Yoshimura N, Oka S, Yoshida M, Hasegawa T. Vascular endothelial growth factor in children with congenital heart disease. Ann Thorac Surg 2003; 75: 15231526. 2. Suda K, Matsumura M, Miyanish S, Uehara K, Sugita T, Matsumoto M. Increased vascular endothelial growth factor in patients with cyanotic congenital heart diseases may not be normalized after a Fontan type operation. Ann Thorac Surg 2004; 78: 942946; discussion 946947. 3. Klagsbrun M, DAmore PA. Vascular endothelial growth factor and its receptors. Cytokine Growth Factor Rev 1996; 7: 259270. 4. Kendall RL, Thomas KA. Inhibition of vascular endothelial cell growth factor activity by an endogenously encoded soluble receptor. Proc Natl Acad Sci USA 1993; 90: 1070510709. 5. Shibuya M. Vascular endothelial growth factor receptor-1 (VEGFR-1/Flt-1): a dual regulator for angiogenesis. Angiogenesis 2006; 9: 225230; discussion 231. 6. Starnes SL, Duncan BW, Kneebone JM, et al. Vascular endothelial growth factor and basic fibroblast growth factor in children with cyanotic congenital heart disease. J Thorac Cardiovasc Surg 2000; 119: 534539. 7. Himeno W, Akagi T, Furui J, et al. Increased angiogenic growth factor in cyanotic congenital heart disease. Pediatr Cardiol 2003; 24: 127132. 8. Webb NJ, Bottomley MJ, Watson CJ, Brenchley PE. Vascular endothelial growth factor (VEGF) is released from platelets during blood clotting: implications for measurement of circulating VEGF levels in clinical disease. Clin Sci (Lond) 1998; 94: 395404. 9. Hormbrey E, Gillespie P, Turner K, et al. A critical review of vascular endothelial growth factor (VEGF) analysis in peripheral blood: is the current literature meaningful? Clin Exp Metastasis 2002; 19: 651663. 10. Arena E, Ferrero S. Pitfalls in the measurement of serum VEGF in children with congenital heart disease. Ann Thorac Surg 2004; 78: 18841885. 11. Hamada H, Ebata R, Higashi K, et al. Serum vascular endothelial growth factor in cyanotic congenital heart disease functionally contributes to endothelial cell kinetics in vitro. Int J Cardiol 2007; 120: 6671.

Conclusion
In contrast to previous studies, we did not observe elevated vascular endothelial growth factor plasma levels in infants with cyanotic congenital cardiac disease. Levels of soluble vascular endothelial growth factor receptor-1, which are not reported in infants

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