Defective angiogenesis in hypoplastic human fetal lungs correlates with nitric oxide synthase deficiency that occurs despite enhanced angiopoietin-2 and VEGF

Abstract

Lung hypoplasia (LH) is a life-threatening congenital abnormality with various causes. It involves vascular bed underdevelopment with abnormal arterial muscularization leading to pulmonary hypertension. Because underlying molecular changes are imperfectly known and sometimes controversial, we determined key factors of angiogenesis along intrauterine development, focusing at the angiopoietin (ANG)/Tie-2 system. Lung specimens from medical terminations of pregnancy (9-37 wk) were used, including LH due to congenital diaphragmatic hernia (CDH) or other causes, and nonpulmonary disease samples were used as controls. ELISA determination indicated little ANG-1 change during pregnancy and no effect of LH, whereas Tie-2 declined similarly between 9 and 37 wk in LH and controls. By contrast, ANG-2 markedly increased in LH from 24 wk, whereas it remained stable in controls. Because VEGF increased also, this was interpreted as an attempt to overcome vascular underdevelopment. Hypothesizing that its inefficiency might be due to impaired downstream mechanism, endothelial nitric oxide synthase (eNOS) was determined by semiquantitative Western blot and found to be reduced by approximately 75%, mostly in the instance of CDH. In conclusion, angiogenesis remains defective in hypoplastic lungs despite reactive enhancement of VEGF and ANG-2 production, which could be due, at least in part, to insufficient eNOS expression.

Figure 1

Representative histological sections of fetal human lungs aged 32–33 weeks (fetal age) without (A, control lung) or with (B, CDH, and C, urogenital defect) lung hypoplasia (LH). Tissue was stained by orcein-picroindigo-carmine. All pictures are at same magnification. Structure of hypoplastic lungs appears immature with dense parenchyma, thicker septa, and reduced presumptive airspaces. Note thickening of blood-vessel walls in B and C.

Figure 2

Immunostaining of platelet endothelial cell adhesion molecule-1 (PECAM-1). Lung sections from fetuses aged 32–33 weeks (fetal age) without (A, control lung) or with (B, CDH, and C, urogenital defect) LH were immunostained with mouse monoclonal anti-human PECAM-1 antibody and counterstained with hematoxylin. All pictures are at same magnification. Control fetuses had extensive staining of endothelial cells within septa, whereas immunostaining appeared less intense in the hypoplastic lungs that displayed reduced complex intertwining of alveolar capillaries.

Figure 3

Scatter plots of ANG1, ANG2, and TIE2 concentrations (pg/mg protein) in human lung fetuses as a function of fetal age (wk). Individual values are shown: open, black, and grey points correspond to control fetuses, fetuses with pulmonary hypoplasia due to CDH, and fetuses with pulmonary hypoplasia due to other causes, respectively. Solid line represents the regression line of control fetuses, hatched line represents the regression line of fetuses with lung hypoplasia (either cause). Two extreme ANG1, ANG2, and TIE2 outliers were discarded for calculating scatters plots. Difference between control and LH fetuses (CDH + other causes) was significant only for Ang2 that was increased in late gestation (LH slope vs Control slope: p= 0.00154). Clinical data of fetuses are depicted in Tables 1 and 2.

Figure 4

Scatter plot of VEGF₁₆₅ concentration (pg/mg protein) in human lung fetuses as a function of fetal age (wk). Individual values are shown: open, black, and grey points correspond to control fetuses, fetuses with pulmonary hypoplasia due to CDH, and fetuses with pulmonary hypoplasia due to other causes, respectively. Solid line represents the regression line of control fetuses, hatched line represents the regression line of fetuses with lung hypoplasia (either cause). VEGF₁₆₅ was markedly increased in hypoplastic lungs in late gestation (LH vs controls slope: p=0.02).

Figure 5

eNOS protein level as a function of developmental stages in human lung fetuses with or without pulmonary hypoplasia. Canalicular stage: (A) representative western blots in CDH, lung hypoplasia due to other causes (LHo), and control lungs; (B) corresponding densitometric analysis normalized by Ponceau. Saccular and alveolar stages: (C) representative western blots; (D) densitometric analysis for saccular stage normalized by Ponceau S. Signal for eNOS protein was evidenced at 135 kDa (B and D). Results are expressed as percentage of control-group mean value. Box plot: the boundary of the box indicates the 25th and 75th percentiles, the bold line and the cross within the box mark the median and mean values, respectively, and whiskers represent minimum and maximum values. eNOS protein was significantly diminished in CDH and LHo groups at the canalicular stage, and in CDH group at the saccular stage (significant difference with respective control group for: (a) p<0.05; (b) p<0.01).