Increased Superoxide Generation Is Associated With Pulmonary Hypertension in Fetal Lambs

Abstract

Abstract-Ligation of the ductus arteriosus in utero produces pulmonary hypertension and vascular remodeling in fetal and newborn lambs. However, the mechanisms producing these vascular changes are not well defined. Because reactive oxygen species (ROS) have been implicated as mediators of smooth muscle cell proliferation, we hypothesized that increased formation of ROS may be involved in the pathophysiology of pulmonary hypertension after in utero ductal ligation. Using ethidium fluorescence, we demonstrated an increase in superoxide levels after 9 days of ductal ligation compared with control lungs (PϽ0.05) that was localized to the adventitia and smooth muscle cells of hypertensive vessels. SOD-1 and SOD-2 protein levels and activities in lung, vein, and artery of hypertensive lambs were unchanged relative to controls after 2 days of ductal ligation. However, after 9 days, superoxide dismutase (SOD) activity was significantly decreased in arteries from ligated lambs without associated changes in SOD protein expression (PϽ0.05).Examination of NADPH oxidase expression as a potential source of the superoxide production indicated that the levels of p67 phox , a subunit of the NADPH oxidase complex, were significantly increased in the pulmonary arteries, but not veins, from the ligated lung as early as 2 days (PϽ0.05). Functional analyses demonstrated that reducing superoxide levels significantly increased the NO-mediated relaxation of pulmonary arteries isolated after 9 days, but not 2 days, of ductal ligation (PϽ0.05). These results suggest that increased NADPH oxidase expression may increase levels of superoxide in persistent pulmonary hypertension of the newborn lung tissue, and that increased superoxide blunts vascular relaxations to exogenous NO while stimulating smooth muscle cell growth. Key Words: reactive oxygen species Ⅲ vascular remodeling Ⅲ smooth muscle W ith initiation of ventilation and oxygenation at birth, pulmonary vascular resistance decreases and pulmonary blood flow increases. 1 In some neonates, this normal transition does not occur, resulting in persistent pulmonary hypertension of the newborn (PPHN). PPHN can occur idiopathically or as a complication of a variety of cardiorespiratory disorders, including hyaline membrane disease, asphyxia, meconium aspiration, and congenital diaphragmatic hernia. 2,3 In newborns that die with PPHN, there is an increase in both the thickness of the smooth muscle layer within small pulmonary arteries and an extension of this muscle to nonmuscular arteries. 4 There is also proliferation of adventitial tissue. 5 These structural changes indicate that in utero events have altered the pulmonary circulation. However, the mechanism underlying these changes is unclear. Surgical ligation or constriction of the ductus arteriosus in the fetal lamb several days before delivery at term results in PPHN after delivery. 6 -9 This lamb model closely resembles the human condition, with suprasystemic pulmonary arterial pressure and anatomic changes including muscularization o...

Role of Oxidized Lipids in Pulmonary Arterial Hypertension

“…In a lamb model of PH of the newborn, it has been shown that the NADPH oxidase enzyme complex may contribute to proliferation of SMCs by producing increasing superoxide levels. 115,116 Moreover, the proliferative effect of endothelin 1 on fetal PASMCs is a consequence of increased generation of ROSs. 117 Regulation of the growth of PASMCs by the transcription factor GATA4 is inhibited by antioxidant serotonin via suppression of NADPH oxidase or monoamine oxidase A, further reinforcing the critical role of ROSs in altering signaling pathways involved in PH.…”

Pivotal Role for Endothelial Tetrahydrobiopterin in Pulmonary Hypertension

“…11,12 Scavenging by superoxide radicals may account in part for reduced NO bioactivity. 13 Increased superoxide production has been observed in experimental models of pulmonary hypertension, 14 and markers of oxida-tive stress are increased in patients with pulmonary hypertension. 15 Superoxide mediates pulmonary vasoconstriction 16 and stimulates pulmonary smooth muscle cell proliferation.…”

“…NOS activity was determined by 14 C-arginine to 14 C-citrulline conversion with HPLC quantification as described. 24 Lung homogenates (400 g protein) were incubated for 30 minutes at 37°C in 500 L Krebs-HEPES buffer containing 14 C-arginine (2 L of 50 Ci/mL), FAD (10 mol/L), FMN (10 mol/L), and NADPH (1 mmol/L).…”

“…Background signals were corrected from samples with 14 C-arginine alone without homogenates. The integrated 14 C-citrulline elution peak was expressed as a percentage of total 14 C counts.…”

Persistent Pulmonary Hypertension of the Newborn: Recent Advances in the Management

“…Superoxide is an oxygen free radical generated in pulmonary arteries by xanthine oxidase, NADPH-oxidase, or uncoupled eNOS in ECs [66]. Although, mechanism of ROS induction is not well-known, endothelin and angiotensin are found to activate NADPHoxidase in vascular SMCs [66][67][68]. Decreased availability of arginine, tetrahydrobiopterin, and heat shock protein-90, lead to uncoupling of eNOS and release of superoxide [68].…”