Postnatal estradiol up-regulates lung nitric oxide synthases and improves lung function in bronchopulmonary dysplasia

Abstract

Rationale: Nitric oxide (NO) plays an important role in lung development and perinatal lung function, and pulmonary NO synthases (NOS) are decreased in bronchopulmonary dysplasia (BPD) following preterm birth. Fetal estradiol levels increase during late gestation and estradiol up-regulates NOS, suggesting that after preterm birth estradiol deprivation causes attenuated lung NOS resulting in impaired pulmonary function.

Objective: To test the effects of postnatal estradiol administration in a primate model of BPD over 14 days after delivery at 125 days of gestation (term = 185 d).

Methods: Cardiopulmonary function was assessed by echocardiography and whole body plethysmography. Lung morphometric and histopathologic analyses were performed, and NOS enzymatic activity and abundance were measured.

Measurements and main results: Estradiol caused an increase in blood pressure and ductus arteriosus closure. Expiratory resistance and lung compliance were also improved, and this occurred before spontaneous ductal closure. Furthermore, both oxygenation and ventilation indices were improved with estradiol, and the changes in lung function and ventilatory support requirements persisted throughout the study period. Whereas estradiol had negligible effect on indicators of lung inflammation and on lung structure assessed after the initial 14 days of ventilatory support, it caused an increase in lung neuronal and endothelial NOS enzymatic activity.

Conclusions: In a primate model of BPD, postnatal estradiol treatment had favorable cardiovascular impact, enhanced pulmonary function, and lowered requirements for ventilatory support in association with an up-regulation of lung NOS. Estradiol may be an efficacious postnatal therapy to improve lung function and outcome in preterm infants.

Figure 1.

(A) Fetal estradiol (E₂) levels increase in the latter half of the third trimester of primate pregnancy. Serum E₂ was measured in fetal baboons at 125 days, 140 days, 160 days, or 180 days gestation upon killing immediately at delivery. Values are mean ± SEM, n = 6/group. *P < 0.05 versus 125 days gestation. (B) Subcutaneous E₂ administration raises serum levels in the immediate postnatal period. The hormone was provided postnatally to preterm baboons delivered by Cesarean section at 125 days gestation by placement of a 0.5 mg, 21-day extended release pellet subcutaneously in the left axilla at 1 hour of life. Control animals received a placebo pellet, and a second E₂ or control pellet was placed subcutaneously in the right axilla on Day 7 of life. Serum levels were determined at 6 hours of life and at 1, 2, 3, 7, 10 and 14 days of age. Open circles = control group; Closed circles = estradiol group. Values are mean ± SEM, n = 8 and 9 for control and E₂-groups, respectively. *P < 0.05 versus control.

Figure 2.

Postnatal estradiol (E₂) administration increases systemic blood pressure (BP) and causes closure of the ductus arteriosus. (A) Systemic mean, systolic and diastolic blood pressures were measured via an arterial catheter. (B) Ductal patency was determined by echocardiography. Green indicates an open ductus, red indicates a closed ductus, and white indicates that an echocardiogram was not performed. Values are mean ± SEM, n = 8 and 9 for control and E₂-groups, respectively. Open circles = control group; Closed circles = estradiol group. Statistical comparisons of BP between groups were made by repeated measures analysis of variance, and ductal patency was compared by two-way analysis of variance. P < 0.05 for mean BP, diastolic BP and ductal patency.

Figure 3.

Postnatal estradiol (E₂) administration causes improvements in pulmonary function. (A) Expiratory resistance (cm H₂O/ml/s) and (B) compliance (ml/cm H₂O/kg) were measured by whole body plethysmography. Open circles = control group; Closed circles = estradiol group. Reported values are for the respiratory system as a whole. Values are mean ± SEM, n = 8 and 9 for control and E₂ groups, respectively. Statistical comparisons were made between groups by two-way ANOVA. P < 0.05 for expiratory resistance and for compliance. Exp. Res. = expiratory resistance.

Figure 4.

Postnatal estradiol (E₂) administration causes improvements in (A) oxygenation index and (B) ventilation index. Open circles = control group; Closed circles = estradiol group. Values are mean ± SEM, n = 8 and 9 for control and E₂ groups, respectively. Statistical comparisons were made between groups by two-way analysis of variance (P < 0.05 for both indices).

Figure 5.

Representative lungs from (A) 125 days gestation, (B) 140 days gestation (C) control group and (D) E₂-treated group. Original magnification, ×20, bar = 100 μm.

Figure 6.

Impact of postnatal estradiol (E₂) administration on elastin distribution and expression. Hart's staining indicated that elastic fibers were localized to both alveolar walls and septal tips (arrows) in lungs of (A) control-treated animals and primarily to emerging septal tips in (B) E₂-treated lungs. In situ hybridization on serial sections similarly detected often intense elastin mRNA expression within alveolar walls and at emerging septae in (C) control group, and expression was localized primarily to emerging septae in (D) E₂-treated animals. Findings in A–D are representative of those in four to five animals per group, original magnification, ×200; bar = 50 μm. (E) Elastin mRNA expression was evaluated by quantitative reverse transcriptase–polymerase chain reaction (RT-PCR). GAPDH = glyceraldehyde 3-phosphate dehydrogenase. Values are mean ± SEM; n = 6/group.

Figure 7.

Postnatal estradiol (E₂) administration up-regulates lung neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) enzymatic activity. Using arginine-to-citrulline conversion, total, calcium-dependent and calcium-independent NOS activity was measured in (A) proximal lung. (B) nNOS-derived and eNOS-derived enzymatic activity was also quantified. (C) nNOS and (D) eNOS protein abundance was evaluate by immunoblot analysis. Open circles = control group; Closed circles = estradiol group. In (C) and (D), upper panels display representative immunoblots for NOS and actin, and lower panels show the cumulative findings for NOS abundance relative to actin in lungs from six animals per group. C = control group; E₂ = estradiol group. Values are mean ± SEM, *P < 0.05 versus control.