Discussion
Angiogenesis and Hypoxia Markers in Heart Tissue of Foetuses With Congenital Heart Defect
This study presents the first evidence of abnormal angiogenesis in heart tissue of human foetuses with CHD compared with normal heart controls which showed increased VEGF-A and sFlt-1 expression and overproduction of proteins such as HIF-2α, HO-1 and SOD1 as a result of chronic hypoxia.
Vascular endothelial growth factor -A expression is dynamic during embryonic heart development and is involved in both long-range and short-range signalling that influences vessel patterning. In mutant mouse embryos, two to three-fold overexpression of VEGF-A resulted in severe abnormalities in heart development, including attenuated compact layer of the myocardium, overproduction of trabeculae, defective ventricular septation, and remodelling of the outflow tract. Interestingly, sFlt-1 expression in our study was increased two-fold in CHD samples compared with controls. Flt-1 has also been implicated in the regulation of embryonic heart function and cardiac morphogenesis.
Placental growth factor has emerged as a central mediator in both coordination of cardiomyocyte growth and neo-angiogenesis. Genetic and pharmacological studies identified PlGF as a novel cardioprotective factor. In our study, the lack of increased PlGF production and overproduction of sFlt-1 and VEGF suggests that CHDs might be caused by the combination of a heart-specific vascular defect and a placental anti-angiogenic environment.
This study also evaluated the presence of hypoxia in abnormal human foetal hearts by analysing the expression of HIF-1-α, HIF-2-α, HO-1, and SOD1. In CHD heart tissue, an overexpression of HIF-2-α and HO-1 was observed, probably as a consequence of chronic hypoxia. Previous in vitro studies demonstrated that HIF-2-α preferentially activates VEGF expression. Some experimental models also showed the importance of HO-1 in heart development. Increased SOD1 expression in CHD cases, as a cell response to tissular hypoxia, would be consistent with the presence of oxidative stress in hearts from CHD foetuses. In our study, the increased expression of hypoxia-inducible genes suggests that a certain degree of hypoxia might be present in CHD. Although chronic hypoxia might up-regulate the expression of pro-angiogenic factors, an overall deregulation of angiogenesis with a net balance towards an anti-angiogenic environment was observed in heart tissue from foetuses with CHD, pointing to an intrinsic angiogenic impairment in these cases. However, as the evaluation of these factors was made in the second trimester of pregnancy, and not during embryogenesis, we are unable to confirm whether angiogenic imbalance leads to abnormal heart development or rather is a consequence of the heart defect.
Angiogenic/Anti-angiogenic Factors in Blood of Women Carrying a Congenital Heart Defect Foetus
The findings of this study showed that, in isolated major foetal heart defects, maternal serum PlGF was decreased and sFlt-1 increased at 18–37 weeks' gestation, suggesting impaired placental angiogenesis. Such impairment was observed in the presence of conotruncal and septal-valve defects but not of left heart defects. In very early foetal mouse development, VEGF-A expression was found in most endocardial cells of the heart tube, whereas at Day 9.5 this expression became restricted to endocardial cells located at points of cushion formation. VEGF-A is required for the activation, proliferation, and remodelling of cushion cells into valve leaflets. Our results appear to show that abnormal angiogenesis may also be deleterious for septal-valve and outflow track formation in human heart embryogenesis.
The aetiologies of left-sided heart malformations are likely complex, with environmental exposures, chromosomal abnormalities, such as Turner's syndrome and Jacobsen's syndrome, and they are strongly determined by genetic factors. A genetic component in the NOTCH1 pathway has been described for patients with LVOT malformations. A family-based study in patients with left congenital cardiac defects identifies both cosegregating and de novo copy number variants enriched in angiogenesis that accounted for up to 10% of left-sided heart disease. The presence of a non-significant trend towards low PlGF levels in cases with LVOT defects in this study might indeed indicate the presence of angiogenic imbalance in a specific subset of these patients, and deserves further investigation.
Several explanations exist for the abnormal angiogenic pattern in maternal blood in pregnant women carrying a foetus with CHD. Our data suggest that these foetuses may have an intrinsic altered angiogenesis leading to abnormal formation of the heart that may also be present in trophoblastic cells. Alternatively, low PlGF may be associated with a lesser degree of trophoblast invasion of the spiral arteries and ensuing placental hypoxia. Placental hypoxia due to abnormal angiogenesis may cause a certain degree of foetal hypoxia that is related to abnormal heart development.
The association of complex CHD, low birth weight, and small head circumference is well established. Disease-specific biometrics are thought to be a consequence of blood flow disturbance to the body caused by abnormal morphology and oxygen distribution in foetuses with heart defects. However, a correlation between lower maternal PlGF levels and birth weight percentile in CHD babies was found in our study, suggesting that placental impairment contributes to diminished growth potential of foetuses with CHD. In addition to low birthweight, foetuses with CHD showed a trend towards a higher umbilical and lower middle cerebral artery Doppler resistance compared with controls. These findings were in line with previous studies that consistently showed that CHD have significantly reduced blood flood resistance in the brain. However, in contrast to common findings in patients with placental complications of pregnancy, uterine Doppler and placental weight were within the normal range in CHD cases. These results are in line with a previous study in the first trimester of pregnancy, reporting that uterine artery Doppler and pregnancy associated plasma protein-A levels in women with a CHD foetus were similar to those of controls. In both this and the current study, clinical cases of placental dysfunction, i.e. intrauterine growth restriction and pre-eclampsia, were excluded, which may explain the absence of clear differences in Doppler and placental parameters among the study groups. We acknowledge that placental histology information might have offered important insights in relation with the interpretation of this potential association, and that future studies including this information are warranted. We believe that evaluating the relationship between CHD and placental-related complications is an important hypothesis to explore as the subject of future research.
Angiogenic/Anti-angiogenic Factors in Cord Blood From Congenital Heart Defect Foetuses
A net anti-angiogenic imbalance with significantly higher sFlt-1 levels and sEng was found in cord blood of CHD compared with control foetuses. Exogenous sFlt-1 alone caused diastolic dysfunction in wild-type mice. Anti-angiogenic therapies, including antibodies that neutralize VEGF and small-molecule VEGF receptor inhibitors, are being increasingly used in the oncological and ophthalmological settings in which cardiomyopathy and heart failure have recently been recognized as major side effects. Peripartum cardiomyopathy, often a fatal disease that affects pregnant women, was recently associated with a systemic angiogenic imbalance. Angiogenic factors play an important role in the development of atherosclerosis and show pronounced changes during acute myocardial infarction (AMI). High sFlt-1 levels proved to be a good predictor of mortality during a 1-year follow-up of AMI, regardless of information provided by troponin T and N-terminal pro-B-type natriuretic peptide. Overall, these studies indicated that an anti-angiogenic state could be harmful to the human heart.
To what extent anti-angiogenic imbalance in the uterus determines cardiac function and neurodevelopment in infants with CHD remains to be determined. Although it is becoming well established that prenatal factors may contribute to perinatal mortality and neurodevelopment morbidity associated with CHD, the specific prenatal causes and mechanisms of insult are largely unknown. The main hypothesis is that major circulatory disorders and ensuing chronic hypoxia may lead to abnormal neurological development in foetuses with CHD; however, our study also revealed a certain degree of placental impairment in these cases which may contribute to a deficient supply of oxygen and metabolic substrate to the developing brain. Moreover, this anti-angiogenic environment during foetal life could adversely impact on the adaptative capabilities of the heart later in life.