Persistent Pulmonary Hypertension Of The Newborn Lambs Research Articles

Milrinone enhances relaxation to prostacyclin and iloprost in pulmonary arteries isolated from lambs with persistent pulmonary hypertension of the newborn

Prostacyclin is a pulmonary vasodilator and is produced by prostacyclin synthase and stimulates adenylate cyclase (AC) via the prostacyclin receptor (IP) to produce cAMP. Forskolin is a direct stimulant of AC. Phosphodiesterase 3 hydrolyzes cAMP and is inhibited by milrinone. To characterize the prostacyclin-AC-cAMP pathway in the ovine ductal ligation model of persistent pulmonary hypertension of the newborn (PPHN). University-based laboratory animal facility. Lambs delivered to time-dated pregnant ewes. Fifth generation pulmonary arteries (PA) and lung parenchyma were isolated from control fetal lambs (n = 8) and fetal lambs with PPHN induced by antenatal ductal ligation (n = 9). We studied relaxation responses to various agonists (milrinone, forskolin, prostacyclin, and iloprost, a prostacyclin analog) that increase cAMP in PA after half-maximal constriction with norepinephrine and pretreatment with propranolol +/- indomethacin. Lung protein levels of prostacyclin synthase, IP, AC2, and phosphodiesterase 3A were analyzed by Western blot and cAMP by enzyme-linked immunoassay. Milrinone relaxed control and PPHN PA and pretreatment with indomethacin significantly impaired this response. Relaxation to milrinone, prostacyclin, and iloprost were significantly impaired in PA from PPHN lambs. Pretreatment with milrinone markedly enhanced relaxation to prostacyclin and iloprost in PPHN PA, similar to relaxation in control PA. Relaxation to forskolin was similar in control and PPHN PAs indicating normal AC activity. Protein levels of prostacyclin synthase and IP were decreased in PPHN lungs compared with control, but AC2, cAMP, and phosphodiesterase 3A remained unchanged. Prostacyclin and iloprost are dilators of PAs from PPHN lambs and their effect is enhanced by milrinone. This combination therapy may be an effective strategy in the management of patients with PPHN.

Superoxide Dismutase Improves Oxygenation and Reduces Oxidation in Neonatal Pulmonary Hypertension

Hyperoxic ventilation in the management of persistent pulmonary hypertension of the newborn (PPHN) can result in the formation of reactive oxygen species, such as superoxide anions, which can inactivate nitric oxide (NO) and cause vasoconstriction and oxidation. To compare the effect of intratracheal recombinant human superoxide dismutase (rhSOD) and/or inhaled NO (iNO) on systemic oxygenation, contractility of pulmonary arteries (PAs), and lung reactive oxygen species (isoprostane, 3-nitrotyrosine) levels in neonatal lambs with PPHN. Six newborn lambs with PPHN (induced by antenatal ductal ligation) were killed at birth. Twenty-six PPHN lambs were ventilated for 24 h with 100% O(2) alone (n = 6) or O(2) combined with rhSOD (5 mg/kg intratracheally) at birth (n = 4), rhSOD at 4 h of age (n = 5), iNO (20 ppm, n = 5), or rhSOD + iNO (n = 6). Contraction responses of fifth-generation PAs to norepinephrine and KCl, lung isoprostane levels, and 3-nitrotyrosine fluorescent intensity were measured. Systemic oxygenation was impaired in PPHN lambs and significantly improved (up to threefold) in both rhSOD groups with or without iNO. Oxygenation improved more rapidly with the combination of rhSOD + iNO compared with either intervention alone. Norepinephrine- and KCl-induced contractions and lung isoprostane levels were significantly increased by 100% O(2) compared with nonventilated newborn lambs with PPHN. Both rhSOD and iNO mitigated the increased PA contraction response and lung isoprostane levels. Intratracheal rhSOD decreased the enhanced lung 3-nitrotyrosine fluorescence observed with iNO therapy. Intratracheal rhSOD and/or iNO rapidly increase oxygenation and reduce both vasoconstriction and oxidation in newborn lambs with PPHN. This has important implications for clinical trials of rhSOD and iNO in newborn infants with PPHN.

Vascular Endothelial Growth Factor Improves Pulmonary Vascular Reactivity and Structure in an Experimental Model of Chronic Pulmonary Hypertension in Fetal Sheep

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by elevated pulmonary vascular resistance, impaired vasodilation, and muscularization of small pulmonary arteries. Past studies have shown that lung vascular endothelial growth factor (VEGF) surges just prior to birth but is markedly decreased in a fetal lamb model of PPHN. In addition, treatment of fetal lambs with an inhibitor of VEGF-165 caused pulmonary hypertension, impaired vasodilation, and increased pulmonary artery muscularization. We hypothesized that treatment with recombinant human VEGF-165 would reduce the severity of pulmonary vascular remodeling and improve endothelial-dependent vasodilation in an experimental model of PPHN. We studied the effects of daily intrapulmonary infusions of recombinant human VEGF in fetal lambs after partial ligation of the ductus arteriosus. We performed surgery in 23 late-gestation fetal lambs (125 to 128 days gestation; term, 147 days), with catheters placed in the main pulmonary artery, left atrium, and aorta for pressure measurements. A catheter was placed in the left pulmonary artery for drug infusions, and a ligature was placed around the ductus arteriosus. Recombinant human VEGF-165 (50 μg/d; n = 11) or its vehicle (n = 12) were infused for 7 or 14 days. In the first group, acetylcholine (15 μg) or 8-bromo-guanosine monophosphate (1.5 mg) were infused on days 2 and 13 to assess endothelium-dependent and endothelium-independent vasodilation, respectively. Animals in the second group had morphometric analysis of small pulmonary arteries performed. We found that although pulmonary vasodilation to 8-bromo-guanosine monophosphate remained intact, acetylcholine-induced vasodilation was reduced in PPHN control lambs after 14 days (change in pulmonary blood flow from baseline, 106% vs 11%; p < 0.05). In contrast, the response to acetylcholine was preserved in lambs treated with recombinant human VEGF (change in pulmonary blood flow, 94% vs 90%; p = not significant). At autopsy, recombinant human VEGF treatment decreased pulmonary artery wall thickness by 34% vs PPHN lambs (p < 0.001 vs ligation). We conclude that VEGF-165 improves endothelium-dependent vasodilation and reduces the severity of pulmonary vascular remodeling in experimental PPHN. We speculate that impaired VEGF signaling contributes to endothelial dysfunction and altered vascular structure in experimental PPHN.

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.

Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased endothelial nitric oxide synthase (eNOS) gene expression in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are elevated in the pulmonary arteries from PPHN lambs and that ET-1 increases ROS production in pulmonary arterial smooth muscle cells (PASMC) in culture. Thus the objective of this study was to determine whether there was a feedback mechanism between the ET-1-mediated increase in ROS in fetal PASMC (FPASMC) and a decrease in eNOS gene expression in fetal pulmonary arterial endothelial cells (FPAEC). Our results indicate that ET-1 increased H2O2 levels in FPASMC in an endothelin A receptor-dependent fashion. This was observed in both FPASMC monoculture and in cocultures of FPASMC and FPAEC. Conversely, ET-1 decreased H2O2 levels in FPAEC monoculture in an endothelin B receptor-dependent fashion. Furthermore, ET-1 decreased eNOS promoter activity by 40% in FPAEC in coculture with FPASMC. Promoter activity was restored in the presence of catalase. In FPAEC in monoculture treated with 0-100 microM H2O2, 12 microM had no effect on eNOS promoter activity, but it increased eNOS protein levels by 50%. However, at 100 microM, H2O2 decreased eNOS promoter activity and protein levels in FPAEC by 79 and 40%, respectively. These data suggest a role for smooth muscle cell-derived H2O2 in ET-1-mediated downregulation of eNOS expression in children born with PPHN.

Pulmonary and systemic effects of the phosphodiesterase inhibitor dipyridamole in newborn lambs with persistent pulmonary hypertension.

Nitric oxide (NO) relaxes vascular smooth muscle by increasing the intracellular concentration of cGMP. In the pulmonary circulation, cGMP is inactivated by specific phosphodiesterases (PDE5). Dipyridamole, a clinically approved drug, has inhibitory activity against PDE5 and has been reported to augment the response to inhaled NO in persistent pulmonary hypertension of the newborn (PPHN). We wished to determine whether dipyridamole alone, or in combination with NO, can be used to treat a newborn lamb model of PPHN. In newborn lambs with PPHN, dipyridamole infused at 0.02 mg/kg/min for 45 min alone, or in combination with 5 ppm of inhaled NO for the final 15 min, significantly decreased pulmonary and systemic blood pressure, decreased pulmonary vascular resistance, and increased pulmonary blood flow. There was no significant difference between the pulmonary vascular effects of 5 ppm NO alone compared with the effects of NO combined with dipyridamole. In control lambs, the 45-min infusion of dipyridamole did not change pulmonary pressure whereas systemic pressure decreased by 28 +/- 3%. These systemic effects in control lambs persisted 90 min after discontinuing the dipyridamole infusion. Systemic arteries isolated from both control and PPHN lambs were significantly more sensitive to dipyridamole than pulmonary arteries. We conclude that dipyridamole has significant hemodynamic effects in both the pulmonary and systemic circulations of newborn lambs with pulmonary hypertension as well as in the systemic circulation of newborn control lambs. The pronounced effects of dipyridamole on the systemic circulation limits its utility as an adjunct to inhaled NO in the treatment of PPHN.

Soluble Guanylate Cyclase (sGC) is Decreased Within Pulmonary Arterial Smooth Muscle of Fetal Lambs with Experimentally-Induced Pulmonary Hypertension.• 1486

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by structural and functional abnormalities of the pulmonary vasculature. Ligation of the ductus arteriosus in fetal lambs 10 days prior to delivery produces an animal model of PPHN. We previously reported that pulmonary arteries isolated from PPHN lambs demonstrate a blunted response to nitric oxide (NO). We now studied lungs isolated from fetal lambs (136 days gestation) using immunohistochemical staining techniques to determine differences in sGC distribution in the pulmonary vasculature between control lambs (n = 4) and lambs with PPHN after ductal ligation (n = 4). Following removal of lungs, barium gelatin was infused into the pulmonary arteries to distinguish small intra-alveolar arteries from post-capillary venules. A B4 primary antibody to the 82 kDa alpha subunit of rat sGC which cross-reacts with sheep sGC was used. Immunoreaction product was measured semiquantitatively using a three point scoring system. Structurally, pulmonary arteries from PPHN lambs displayed increased musculature of the medial layer and thickening of the adventitia. When compared to controls, sGC immunostaining in lambs with PPHN was significantly depressed at all levels of the arterial tree. The largest decrease in immunostaining compared to controls was seen at the level of the terminal bronchiole followed by arteries at the level of bronchi. In the venous tree, sGC immunostaining in PPHN lambs appeared unchanged from controls with the exception of postcapillary venules in which staining was significantly increased. These findings support our previous functional studies, and suggest there is decreased expression of sGC within vascular smooth muscle cells in this model of PPHN.