Amy N. MacRitchie

Estrogen Upregulates Endothelial Nitric Oxide Synthase Gene Expression in Fetal Pulmonary Artery Endothelium

NO, produced by endothelial NO synthase (eNOS), is a key mediator of pulmonary vasodilation during cardiopulmonary transition at birth. The capacity for NO production is maximal at term because pulmonary eNOS expression increases during late gestation. Since fetal estrogen levels rise markedly during late gestation and there is indirect evidence that the hormone enhances nonpulmonary NO production in adults, estrogen may upregulate eNOS in fetal pulmonary artery endothelium. Therefore, we studied the direct effects of estrogen on eNOS expression in ovine fetal pulmonary artery endothelial cells (PAECs). Estradiol-17beta caused a 2.5-fold increase in NOS enzymatic activity in PAEC lysates. This effect was evident after 48 hours, and it occurred in response to physiological concentrations of the hormone (10(-10) to 10(-6) mol/L). The increase in NOS activity was related to an upregulation in eNOS protein expression, and eNOS mRNA abundance was also enhanced. Estrogen receptor antagonism with ICI 182,780 completely inhibited estrogen-mediated eNOS upregulation, indicating that estrogen receptor activation is necessary for this response. In addition, immunocytochemistry revealed that fetal PAECs express estrogen receptor protein. Furthermore, transient transfection assays with a specific estrogen-responsive reporter system have demonstrated that the endothelial estrogen receptor is capable of estrogen-induced transcriptional transactivation. Thus, estrogen upregulates eNOS gene expression in fetal PAECs through the activation of PAEC estrogen receptors. This mechanism may be responsible for pulmonary eNOS upregulation during late gestation, thereby optimizing the capacity for NO-mediated pulmonary vasodilation at birth.

Ontogeny of cyclooxygenase-1 and cyclooxygenase-2 gene expression in ovine lung

Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.Prostacyclin is a key mediator of pulmonary vascular and parenchymal function during late fetal and early postnatal life, and its synthesis in whole lung increases during that period. The rate-limiting enzyme in prostacyclin synthesis in the developing lung is cyclooxygenase (COX). We investigated the ontogeny and cellular localization of COX-1 (constitutive) and COX-2 (inducible) gene expression in lungs from late-gestation fetal lambs, 1-wk-old newborn lambs (NB1), and 1- to 4-mo-old newborn lambs (NB2). COX-1 mRNA abundance rose progressively from fetal to NB1 to NB2, increasing 12-fold overall. In parallel, immunoblot analysis revealed a progressive increase in COX-1 protein, rising fourfold from fetal lambs to NB2. COX-2 mRNA levels increased fivefold from fetal to NB1 but were similar in NB1 and NB2. However, COX-2 protein was not detectable by immunoblot analysis in any age group. Immunohistochemistry for COX-1 showed intense immunostaining in endothelial cells at all ages. COX-1 was also expressed in airway epithelium at all ages, with a greater number of epithelial cells staining positively in NB2 compared with fetal and NB1 groups. In addition, COX-1 was expressed in airway smooth muscle from NB1. COX-2 immunostaining was absent in all age groups. These findings indicate that there is differential expression of COX-1 and COX-2 in the developing lung and that the enzymes are expressed in a cell-specific manner. The developmental upregulation in COX-1 may optimize the capacity for prostaglandin-mediated vasodilation, bronchodilation, and surfactant synthesis in the newborn lung.

Nitric Oxide Inhalation Decreases Pulmonary Vascular Resistance in Preterm Lambs With Evolving Chronic Lung Disease. • 1470

Nitric Oxide Inhalation Decreases Pulmonary Vascular Resistance in Preterm Lambs With Evolving Chronic Lung Disease. • 1470

Regulation of Endothelial Nitric Oxide Synthase Expression in Small Pulmonary Arteries and Airways of Chronically Ventilated Preterm Lambs. † 1700

Regulation of Endothelial Nitric Oxide Synthase Expression in Small Pulmonary Arteries and Airways of Chronically Ventilated Preterm Lambs. † 1700

Continuous Inhalation of Nitric Oxide From Birth Preserves the Pulmonary Vasoconstrictor Response to Hypoxia in Chronically Ventilated Preterm Lambs|[bull]| 1614

Chronic lung injury from prolonged mechanical ventilation of preterm lambs inhibits the normal postnatal decrease in pulmonary vascular resistance (PVR) and blunts the vasoconstrictor response to hypoxia (Bland et al, Pediatr Res 39:327A, 1996). These abnormalities of lung vascular tone and reactivity may contribute to impaired respiratory gas exchange that often exists in infants with chronic lung disease (CLD). We previously reported that nitric oxide (NO) inhalation for 1 h reduced PVR in preterm lambs with evolving CLD. To see if continuous NO inhalation from birth might inhibit the pulmonary vascular dysfunction that occurs in this model of CLD, we studied 4 preterm lambs (124± 3 d gestation; term = 147 d) that were mechanically ventilated for 3-4 wks at a respirator rate of 20 breaths/min, tidal volume 10-15 ml/kg, with 5-15 ppm NO and sufficient O2 to keep the PaO2 ≅ 50-90 mmHg. All lambs received surfactant at birth and had surgery for ductus arteriosus ligation and catheter placement to allow weekly assessment of PVR from measurements of pulmonary arterial and left atrial pressures and pulmonary blood flow before and during steady-state hypoxia. Results of these studies were compared with those of similar studies done with 10 lambs of the same gestation that were mechanically ventilated the same way but without NO. After 3 wks of mechanical ventilation, baseline PVR was not significantly different in the 4 lambs that received NO compared with the 10 lambs that did not receive NO (PVR = 44.8 ± 5.4 vs 53.9 ± 21.2 mmHg/(L/sec)/kg, respectively). However, in response to hypoxia (baseline FiO2.28±.09, PaO2 73 ± 3; hypoxia FiO2.17 ±.03, PaO2 35 ± 1), PVR consistently increased from 48.3 ± 2.3 to 74.0 ± 20.0 mmHg/(L/sec)/kg at the end of wk 3 in lambs that received NO from birth, whereas PVR did not increase significantly at the end of wk 3 in chronically ventilated lambs that did not receive NO. When inhaled NO was discontinued for 2-4 h after 3 wks of mechanical ventilation in the 4 NO-treated lambs, PVR increased on average from 50.5 ± 11.7 to 64.5± 18.7 mmHg/(L/sec)/kg. Subsequent hypoxia (FiO2.17 ±.03, PaO2 35 ± 4 mmHg), without inhaled NO, further increased PVR to 87.8 ± 27.2 mmHg/(L/sec)/kg. Thus, continuous nitric oxide inhalation from birth may help to preserve the normal pulmonary vasoconstrictor response to hypoxia and thereby facilitate ventilation-perfusion matching and respiratory gas exchange in chronically ventilated preterm lambs.

Nitric Oxide Upregulates Endothelial Nitric Oxide Synthase Expression in Fetal Pulmonary Artery Endothelial Cells • 1783

Pulmonary vasodilation during cardiopulmonary transition at birth is mediated by endothelium derived nitric oxide (NO), which is generated by the endothelial isoform of NO synthase (NOS), or eNOS. Inhaled NO therapy is currently often used to treat neonatal pulmonary hypertension. Some patients receiving inhaled NO are difficult to wean from the gas after prolonged treatment, suggesting that long-term NO exposure modifies the function of the NO-cGMP signalling cascade. Although NO is known to acutely and reversibly inhibit eNOS activity, the effects of prolonged NO exposure on eNOS expression are not known. We therefore determined the effects of long-term NO exposure on eNOS expression in early passage ovine fetal pulmonary artery endothelial cells (PAEC). To first examine the effects of endogenous NO, PAEC were treated with 2 mM nitro-L-arginine methyl ester (L-NAME). L-NAME caused a decrease in eNOS protein expression that was evident within 8h and maximal by 16h (67% decrease); the effect persisted for at least 48h. The effects of exogenous NO were examined in PAEC treated with the NO donor spermine NONOate or the control parent compound spermine (10-8M to 10-6M). Exogenous NO caused increases in both eNOS protein and cell lysate NOS activity that were also evident within 8h, maximal at 16h (223% of control), and persistent for at least 48h. However, eNOS mRNA abundance was not altered by changes in the level of endogenous or exogenous NO, indicating that the effect of NO on eNOS expression is mediated at the level of translation. This effect is more rapid than previously demonstrated transcriptional regulation of PAEC eNOS by oxygen(≥ 24h). Thus, both endogenous and exogenous NO cause positive feedback, translational upregulation of eNOS gene expression in fetal PAEC. To our knowledge, this is the first demonstration of eNOS regulation mediated at the level of translation. Furthermore, these findings suggest that difficulties with prolonged requirements for inhaled NO in certain patients are not due to changes in the capacity for endogenous NO production.

ENDOTHELIAL NITRIC OXIDE SYNTHASE EXPRESSION IS DECREASED IN CHRONIC LUNG INJURY IN PRETERM LAMBS. † 2016

ENDOTHELIAL NITRIC OXIDE SYNTHASE EXPRESSION IS DECREASED IN CHRONIC LUNG INJURY IN PRETERM LAMBS. † 2016