Pulmonary Development in Congenital Diaphragmetic Hernia

When congenital diaphragmatic hernia (CDH) was first described in the early 18th century, it was considered as a result of an opening in the diaphragm that theoretically could be easily corrected after birth by removal of the herniated viscera and subsequent closure of the diaphragm. Over the past three decades, however, it has become evident that CDH is an anomaly characterized by not only a diaphragmatic defect, but also a variable amount of pulmonary hypoplasia (PH) and lung immaturity in some cases. Apart from these features, pulmonary vascular abnormalities may occur and cause persistent pulmonary hypertension of newborn (PPHN).1,2 Hypoplastic lungs are characterized by fewer airways and smaller air spaces, and thus the lower number of vascular generations and increased adventitia and medial thickness of pulmonary arterial walls give rise to pulmonary hypertension.1,2 Apart from the morphologic abnormalities in the pulmonary vasculature, an alternate expression of various cellular mediators, such as nitric oxide, endothelin, prostaglandins, catecholamines, and renin-angiotensin system, have been suggested to contribute to the pathogenesis of PPHN in CDH patients.3,4 Classically, CDH is considered as a primary defect of diaphragmatic embryogenesis resulting from failure of formation or fusion of pleuroperitoneal membranes. Consequently, the abdominal organs herniate into the ipsilateral thoracic cavity when the midgut returns to the abdominal cavity around the 10th gestational week. The timing of herniation coincides with the critical period of lung development and in utero competition for space of the developing lung and the 'abdominal viscera' leads to pulmonary hypoplasia.5 However, more recent experimental evidence suggests that lung hypoplasia may occur independently as a result of defects in signaling pathways.6 Recently the retinoid hypothesis has gained much interest – reviewed by Clugston et al.7 – and also we have gained much greater understanding of the role of specific genes in the etiology of CDH.8 Different animal models have been used to study the natural history of CDH, but current understanding of its etiology and pathophysiology remains limited

Effect of oxygen on the expression of hypoxia-inducible factors in human fetal lung explants

Background: Fetal lung development requires proper coordination between lung epithelial and vascular morphogenesis. A major determinant in lung vascular development is vascular endothelial growth factor (VEGF), which is regulated by hypoxia-inducible factors (HIFs). VEGF is expressed in the airway epithelium, while its receptors (VEGFRs) are expressed in the pulmonary mesenchyme. The hypoxic environment in utero is beneficial for fetal organogenesis, especially vascular development. However, little is known about the expression of HIFs and VEGFR-2 in the human fetal lung in vitro. Objectives: The purpose of this study was to investigate the effects of hypoxia on fetal lung morphology and mRNA expression of VEGF, VEGFR-2, HIF-2α, and HIF-3α. Methods: An explant culture technique was used to study the effects of normoxic and hypoxic conditions on human fetal lung. Results: The morphology remained largely unchanged in explants cultured under hypoxic or normoxic conditions. Quantitative RT-PCR showed that the mRNA expression of VEGF-A, but not VEGFR-2 is upregulated in explants cultured at 1.5% compared with 21% oxygen. We observed a nonsignificant increase in HIF-2α and HIF-3α mRNA expression in explants cultured at 1.5% oxygen. These data suggest that the mRNA expression of VEGF, and possibly HIF-2α and HIF-3α, is regulated by hypoxia in the developing human lung. Conclusion: This lung explant culture model appears to be a valuable model to unravel the molecular mechanisms of human lung development

Expression of Hypoxia-Inducible Factors, Regulators, and Target Genes in Congenital Diaphragmatic Hernia Patients

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel-Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1 alpha, HIF-2 alpha, HIF-3 alpha, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2 alpha might indicate a role for these factors in the pathophysiology of CDH

Expression of hypoxia-inducible factors in normal human lung development

Pulmonary vascular development is essential for proper lung development, and its disturbance can lead to neonatal morbidity and mortality, as exemplified in congenital diaphragmatic hernia. Hypoxia-inducible factors (HIFs) appear to be key molecules in physiologic angiogenesis and in certain forms of lung pathology, such as bronchopulmonary dysplasia. Little is known about the qualitative and quantitative expression of HIFs in normal human fetal lung development. Therefore, we investigated the expression of HIT-1 alpha, HIF-2 alpha, and HIF-3 alpha, along with their upstream regulators and downstream targets, von Hippel-Lindau protein, vascular endothelial growth factor A (VEGF-A), and its receptor, VEGFR-2, in 20 normal human fetal lungs (13.5 weeks in gestation until term) and 5 adult lungs. Quantitative polymerase chain reaction demonstrated a positive correlation between HIF-2 alpha and VEGF-A expression and gestational age. Although there appeared to be a decreasing trend in HIF-3 alpha expression during pregnancy, it did not reach statistical significance. Immunohistochemistry for HIF-1 alpha and HIF-2 alpha revealed that HIF-1 alpha is expressed in the epithelium, while HIF-2 alpha is expressed in both interstitium and epithelium. Our data indicate that HlFs, most notably HIF-2 alpha, appear to exert an important role in angiogenesis during human fetal lung development, especially in the last phases of pregnancy, preparing the fetus for extrauterine life. As such, our results form the baseline data for the evaluation and interpretation of abnormal pulmonary vascular development

Expression of hypoxia-inducible factors, regulators, and target genes in congenital diaphragmatic hernia patients

Congenital diaphragmatic hernia (CDH) is associated with lung hypoplasia and pulmonary hypertension and has high morbidity and mortality rates. The cause and pathophysiology of CDH are not fully understood. However, impaired angiogenesis appears to play an important role in the pathophysiology of CDH. Therefore, we examined different components of an important pathway in angiogenesis: hypoxia-inducible factors (HIFs); HIF regulators von Hippel - Lindau (VHL) and prolyl 3-hydroxylase (PHD3); and HIF target genes vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR-2). Quantitative polymerase chain reaction of lung tissue showed a significantly decreased expression of VEGF-A mRNA in the alveolar stage of lung development in CDH patients compared with matched control patients. In the canalicular stage, no differences for VEGF-A were seen between the lungs of CDH patients and those of control patients. Other components of angiogenesis (VHL, HIF-1α, HIF-2α, HIF-3α, VEGFR-2 mRNA, PHD3 protein) that were analyzed showed no differences in expression between CDH and control patients, independent of the developmental stage. A lower expression of VEGF mRNA in CDH patients in the alveolar stage, possibly as a result of downregulation of HIF-2α might indicate a role for these factors in the pathophysiology of CDH