Reduced endothelial nitric oxide synthase in lungs of chronically ventilated preterm lambs.

Nitric oxide (NO), produced in lung vascular endothelium and airway epithelium, has an important role in regulating smooth muscle cell growth and tone. Chronic lung disease, a frequent complication of premature birth, is characterized by excess abundance, tone, and reactivity of smooth muscle in the pulmonary circulation and conducting airways, leading to increased lung vascular and airway resistance. Whether these structural and functional changes are associated with diminished pulmonary expression of endothelial nitric oxide synthase (eNOS) protein is unknown. Both quantitative immunoblot analysis and semiquantitative immunohistochemistry showed that there was less eNOS protein in the endothelium of small intrapulmonary arteries and epithelium of small airways of preterm lambs that were mechanically ventilated for 3 wk compared with control lambs born at term. No significant differences were detected for other proteins (inducible NOS, alpha-smooth muscle actin, and pancytokeratin). Lung vascular and respiratory tract resistances were greater in the chronically ventilated preterm lambs compared with control term lambs. These results support the notion that decreased eNOS in the pulmonary circulation and respiratory tract of preterm lambs may contribute to the pathophysiology of chronic lung disease.

Nitric oxide (NO) upregulates NO synthase expression in fetal intrapulmonary artery endothelial cells.

Endothelium-derived nitric oxide (NO) generated by endothelial NO synthase (eNOS) is critically involved in pulmonary vasodilation during cardiopulmonary transition at birth. Inhaled NO therapy has recently been considered for patients with persistent pulmonary hypertension of the newborn (PPHN). To better understand the mechanisms regulating NO synthesis in the developing pulmonary circulation and the possible ramifications of NO therapy, studies were performed with early passage ovine fetal intrapulmonary artery endothelial cells (PAEC) to determine whether NO directly modulates eNOS expression. To examine the effects of exogenous NO, PAEC were treated with the NO donor spermine NONOate or the parent compound spermine. Exogenous NO caused increases in eNOS protein expression and NOS enzymatic activity that were detectable within 16 h of exposure. In contrast, the inhibition of endogenous NO production with nitro-L-arginine-methyl ester (L-NAME) caused a reduction in eNOS protein expression that was evident within 8 h. Paralleling the changes in eNOS protein, eNOS messenger RNA (mRNA) abundance was upregulated by exogenous NO and downregulated by L-NAME, suggesting that NO modulation of eNOS expression involves processes at the level of gene transcription or mRNA stability. Thus, in fetal PAEC there is positive-feedback regulation of eNOS expression by both exogenous and endogenous NO. These findings suggest that difficulties with transient effectiveness or prolonged requirements for NO therapy in certain PPHN patients are not due to declines in eNOS expression. Further, conditions such as fetal hypoxemia that impair PAEC NO production may attenuate eNOS expression through this mechanism, thereby contributing to the pathogenesis of PPHN.

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.

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.