In this longitudinal cohort of infants with CDH, which is the largest prospective study in this patient population to date, we found that ET1 levels at 2 weeks of age are significantly associated with patient outcome at hospital discharge. In addition, ET1 levels are associated with the degree of elevation of Ppa in infants with CDH, by echocardiographic estimates. Thus, our data suggest that plasma ET1 levels are an indicator of disease severity in CDH, although the pathobiology of ET1 in CDH remains undetermined.
In our cohort, almost all infants who are ultimately discharged on room air have transitioned to a lower estimate of Ppa by 2 weeks of age. Our findings at 2 weeks of age suggest that lung and vascular injury attenuate or delay the expected decrease in Ppa seen in almost all infants who are discharged on room air. Thus, the first 2 weeks of life in these infants may be a critical time for changes in the lung and pulmonary vascular bed, which affect later growth and function of the lung. These findings are consistent with data from animal models, which demonstrate a critical period of lung growth during which more severe pulmonary vascular injury will result in irrecoverable arrest of growth and development (20
In infants with CDH, vascular injury likely occurs secondary to abnormal pulmonary blood flow patterns. Prolonged elevations in PVR, a hypoplastic pulmonary vascular bed, and chronic increased blood flow to the lung contralateral to the hernia are all likely factors in this process, along with biochemical abnormalities in NO signaling demonstrated with vascular remodeling (4
). Thus, elevations in ET1 at 2 weeks of age in those infants who have a poor outcome (expired or discharged on oxygen) may reflect differences that are both biochemical (affecting endothelial and vascular smooth muscle cell function) and structural.
We found that ET1 levels in patients with poor outcome tended to increase at 1 to 2 weeks of age and then decrease by 4 weeks among patients who were alive at that point. We also found that ET1 levels were associated with the severity of PH, after adjusting for the timing of the sample. Prior studies using a similar ET1 assay have shown that newborns with PPHN have elevated ET1 levels compared with unaffected control subjects (31 ± 4.7 vs. 15.1 ± 4.1 pg/ml) (30
). Findings in other patient populations may help explain the elevations in ET1 seen in our patients with poor outcome. Increased pulmonary ET1 production (46
) and plasma ET1 levels are demonstrated in adults with PH (with the degree of elevation correlated with the degree of hypertension) (48
). In healthy adults, prior studies have demonstrated either no transpulmonary gradient or a decrease in ET1 levels across the pulmonary circulation, which is the site of clearance of almost half of circulating ET1 (48
). In PH, ET1 concentration may increase across the pulmonary circulation, implicating increased lung ET1 production or decreased clearance in the disorder (26
). Normalization of the ET1 transpulmonary gradient after chronic therapy suggests that these changes in clearance or production can be reversible (51
). One mechanism to explain changes in lung ET1 clearance could be alterations in ETB receptor expression. ETB receptors localized to the endothelium are primarily responsible for lung ET1 clearance (52
). Aberrant expression of the ETB receptor has been demonstrated in the vascular smooth muscle layer in adults with PH (53
) and infants with CDH and PH (32
). Lambs with PH and abnormal reactivity have similar findings (54
), but rats exposed to chronic exogenous ET1 resolve aberrant ETB receptor expression while abnormal vascular reactivity persists (55
). Thus, in our patients, changes in pulmonary clearance or production of ET1 could explain the fall in plasma ET1 levels we observed. Our later results from cardiac catheterization, at 1.5 to 4 months of age, demonstrate no definitive pattern for transpulmonary handling of ET1 and generally lower ET1 levels than we found at 4 weeks.
Increased local or circulating ET1 may be a direct effector of the lung and vascular abnormalities seen in CDH (decreased and dysplastic growth of the lung and its vasculature) rather than just a marker of abnormal endothelial or vascular smooth muscle function. ET1 could be acting as a profibrotic factor, a smooth muscle cell mitogen, or via interactions with the NO-cGMP signaling system. Structural changes are seen in a transgenic mouse model with pulmonary overexpression of ET1, resulting in peribronchial and perivascular fibrosis, with no evidence of PH and normal alveoli (56
). ET1 acts as a vascular smooth muscle cell mitogen via production of reactive oxygen species (ROS) (57
), and ROS production may also result in decreased pulmonary NO bioavailability. Excessive production of ROS is implicated in the pathophysiology of vascular remodeling demonstrated in ovine models of PH that are associated with impairment of NO signaling (58
). Further, the addition of N-acetyl cysteine improves the impaired endothelium-dependent and -independent vasodilation seen in adult rats receiving a chronic infusion of ET1 (55
). We have previously demonstrated abnormal vascular reactivity in infants and children with CDH (60
). Thus, there are potential implications of these findings for treatment in CDH. Therapies focused on enhancing NO bioavailability or modulating the ET1 system may be effective in preventing these biochemical changes or in reversing them once they have occurred. By this mechanism, therapy could result in enhanced lung and vascular growth, which is seen in newborn animal models of PH and lung injury (13
Chronic iNO resulted in increased plasma ET1 levels in these patients only after 9 days of age (with most patients treated continuously from Day 1). Increased lung and circulating ET1 has been described with NO inhalation in animals with normal pulmonary vasculature and in children and adults with cardiopulmonary disease (62
). In human newborns with PPHN, treatment with iNO resulted in lower ET1 levels compared with placebo-treated infants after weaning off therapy, with no differences noted at 1 day of treatment (17
). Interestingly, although plasma ET1 levels are elevated in ARDS (47
), increased respiratory insufficiency as indicated by the need for ongoing assisted ventilation was not independently associated with ET1 levels in our cohort.
The strength of this study is that the children had a spectrum of acute outcomes for newborns presenting at birth with CDH. The combined outcome of death or discharge on oxygen identifies the most severely affected group of infants, and more than half of the children in this study had this poor outcome. Our study is limited by the difficulty in obtaining accurate Ppa estimates by echocardiography, despite the prospective collection of these data. Due to the lack of ductus arteriosus shunt or measureable TR jet, we confined our classification to the relationship of estimated Ppa to systemic pressure, which may have limited our ability to document significant associations. Regardless, previous studies have shown that TR jet in infants with chronic lung disease does not accurately estimate the systolic Ppa obtained at cardiac catheterization, and echocardiography has limited sensitivity and specificity for the severity of PH in this population (43
). Also, we did not control conditions at the time of each echocardiogram, although these patients were treated under a consistent clinical protocol, adding additional variability to our estimates.
In conclusion, this study demonstrates that plasma ET1 levels are significantly associated with disease severity in infants with CDH. Although permissive ventilation strategies have improved survival in these patients, there is still significant mortality and morbidity in this population, with ongoing symptomatic lung dysplasia in survivors. Animal models have demonstrated the importance of dysregulation of the ET1 system and its interaction with the NO signaling cascade in the pathobiology of PH in the developing lung. Thus, therapies that modulate the ET1 system or its effectors (decreased NO signaling or excessive oxidative stress) in infants with severe CDH may impact lung and vascular growth and remodeling in this patient population and improve outcomes.