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Rationale: 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.
Objective: 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.
Methods: 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% O2 alone (n = 6) or O2 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.
Results: 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% O2 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.
Conclusion: 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.
Hyperoxic ventilation causes oxidant injury and can contribute to worsening of pulmonary hypertension in newborns.
Intratracheal recombinant human superoxide dismutase improves oxygenation and reduces pulmonary arterial contractility and oxidation (isoprostane levels) in an ovine model of neonatal pulmonary hypertension
Persistent pulmonary hypertension of the newborn (PPHN) is a serious disorder of newborn infants characterized by a failure to normally decrease pulmonary vascular resistance at birth, and it has a reported incidence of 1.9 per 1,000 live births (1). Infants with PPHN are initially managed with mechanical ventilation with high levels of inspired oxygen and inhaled nitric oxide (iNO), which is ineffective in approximately 40% of infants with PPHN (2, 3).
Hyperoxic ventilation can be life-saving in PPHN, but toxic to the developing lung (4) by the formation of reactive oxygen species (ROS), such as superoxide anions. ROS can react with arachidonic acid, leading to the formation of isoprostanes, which are potent constrictors in pulmonary arteries (PAs) in newborn rats and piglets (5). Superoxide anions also inactivate NO and in the process form peroxynitrite, a potent oxidant with the potential to produce vasoconstriction, cytotoxicity, and damage to surfactant proteins and lipids (6, 7) and nitration of tyrosine residues to form 3-nitrotyrosine (8, 9). Enhanced lung nitrotyrosine staining has been observed in infants dying of respiratory failure (10). Newborn rat PAs exposed to peroxynitrite increase isoprostane production by 10-fold, implicating these products as possible mediators of peroxynitrite-induced vascular constriction (11).
Several lines of evidence indicate that ROS are important in the pathogenesis of PPHN. First, an increase in superoxide anions in the smooth muscle and adventitia of PAs and a reduction in superoxide dismutase (SOD) activity have been demonstrated in the ovine ductal ligation model of PPHN (12). Next, it has been suggested that ROS may have a role in vascular smooth muscle cell proliferation in PPHN (13). In addition, SOD pretreatment of PAs isolated from PPHN lambs enhances the relaxation response to exogenous NO donors (14). Finally, we have previously reported that a 5-mg/kg dose of recombinant human Cu Zn-SOD (rhSOD) administered intratracheally selectively decreased PA pressure and enhanced the pulmonary vasodilator effects of NO at 5 and 80 ppm over 30 min (14).
The objective of the current study was to evaluate the long-term (24-h) effects of intratracheal rhSOD administered either at birth or at 4 h of age with or without iNO in lambs with PPHN. Physiologic responses (oxygenation), ROS formation (lung 8-isoprostane F2α levels and 3-nitrotyrosine immunofluorescence), and the contractile responses of isolated PAs to exogenous vasoconstrictors were used to analyze the efficacy of these treatments compared with newborn lambs with PPHN or PPHN lambs ventilated with 100% oxygen alone for 24 h. We hypothesized that intratracheal rhSOD would improve oxygenation and reduce oxidant injury and PA contractility in lambs with PPHN. Because the safety profile of rhSOD has been established in newborn human infants (15–17), we anticipate that results demonstrating significant benefits of intratracheal rhSOD will have important implications in clinical studies in newborn infants with PPHN, as suggested in a recent review (4). Previously reported preliminary studies have now been further developed and presented in a more final form in the current article (18–21).
The Laboratory Animal Care Committee at the State University of New York (SUNY) Buffalo approved this study. Fetal ductal ligation was performed at 125 d of gestation (term = 145 d) in time-dated pregnant sheep (Swartz family farm, Attica, NY) as previously described (22, 23). After a 9-d recovery, 35 lambs with PPHN were delivered by cesarean section. Three lambs were severely hydropic and were excluded from the study. Six control lambs were killed before their first breath. The remaining 26 lambs were intubated, and administered calfactant 3 ml/kg (Infasurf; ONY, Inc., Amherst, NY). The umbilical cord was clamped and cut, and the lamb was placed under a radiant warmer and ventilated with a Servo 300 ventilator (Siemens, Mississauga, ON, Canada) with the following initial settings: positive end-expiratory pressure, 4 cmH2O; positive inspiratory pressure (PIP), 20 to 25 cm H2O (targeted to provide a tidal volume of 10 ml/kg); and rate, 40 breaths/min. Umbilical arterial and venous lines were placed, and ventilator settings were adjusted to maintain PaCO2 between 35 and 50 mm Hg as described previously (24). Briefly, abnormal PaCO2 values were managed by adjusting the PIP to maintain a tidal volume of 10 ml/kg and/or change in the ventilator rate by 5 breaths/min. Intravenous fluids (dextrose 10% solution with 25 mEq of NaCl, 20 mEq of KCl, 10 mEq of NaHCO3/L) were administered continuously at 100 ml/kg/d. Fluid composition and rate were adjusted based on serum electrolyte values. Lambs were sedated with fentanyl (2 μg/kg/dose every 2 h as needed) and paralyzed with pancuronium bromide (0.1 mg/kg/dose every 4 h as needed). Lambs with hypotension (mean blood pressure < 40 mm Hg) and tachycardia (heart rate > 200 beats/min) received 10 ml/kg of whole blood (either collected from the placenta or the mother). A second transfusion was repeated if these signs persisted. If hypotension persisted despite two blood transfusions; dopamine was started at 5 μg/kg/min and titrated based on the therapeutic response. Thoracotomy and instrumentation of the PAs were not performed. Oxygenation was measured as arterial to alveolar oxygen ratios (a/A ratio = umbilical arterial PaO2 ÷ [745 − 47] × FiO2 − PaCO2/0.8, where 745 mm Hg is the barometric pressure at SUNY Buffalo, and 47 mm Hg is the partial pressure of water).
Lambs were randomized before delivery to receive 100% oxygen and mechanical ventilation alone (n = 6) or 100% oxygen in combination with iNO at 20 ppm continuously through an INOvent (n = 5; INO Therapeutics, Clinton, NJ), intratracheal rhSOD 5 mg/kg (1 mg = 3,850 U of activity; Biotechnology General, Iselin, NJ) at birth (n = 4), or intratracheal rhSOD 5 mg/kg at 4 h of age (n = 5), or rhSOD + iNO at birth (n = 6). The FiO2 in the inspiratory limb of the ventilator circuit was continuously measured with the INOvent and kept constant at close to 1.0. After 24 h of ventilation, the lambs were anesthetized with 10 mg/kg of pentothal and killed by rapid exsanguination.
Isolated fifth-generation PA rings were suspended in aerated (94% O2/6% CO2) modified Krebs-Ringer solution (24, 25). Some PAs isolated from newborn lambs with PPHN were studied in 20% O2/6% CO2 to evaluate the effect of in vitro oxygen exposure on contractile response to norepinephrine (NE) and KCl. Please see online supplement for further details.
Please see online supplement for details.
Please see online supplement for details.
The birth weight, sex, multiplicity, and first blood gas analysis of the ventilated lambs with PPHN are shown in Table 1. All lambs were studied at 134 to 135 d gestational age. There were no significant differences among the groups. The first blood gas was obtained after insertion of the umbilical arterial line (19 ± 6 min of life). There was a tendency towards higher PaO2 values in the lambs that received iNO but this did not reach statistical significance. However, the oxygenation index (OI = mean airway pressure × inspired oxygen concentration ÷ umbilical arterial PaO2) was significantly lower in the 100% O2 + rhSOD + iNO group compared with both 100% O2 + rhSOD treatment groups.
Eleven lambs in this study were hypotensive and required blood transfusion. Three lambs in the 100% O2–only and 100% O2 + iNO + rhSOD groups, two lambs each in the 100% O2 + rhSOD and 100% O2 + iNO groups, and one lamb in the 100% O2 + delayed rhSOD group received blood transfusion. One lamb in the 100% O2 + iNO group and two lambs in the 100% O2–only group had persistent hypotension requiring a dopamine infusion (peak dose, 15 μg/kg/min).
Figures 1 and and22 show arterial oxygenation in the five groups of lambs expressed as a/A ratios. All lambs were hypoxic with an a/A ratio of 0.038 ± 0.01 and a PaO2 of 40.2 ± 5.1 mm Hg on the first postnatal arterial blood gas in approximately 100% O2. As expected, PPHN lambs ventilated with 100% O2 remained critically ill, with a mean a/A ratio at 24 h of 0.19 ± 0.08 (control lambs without PPHN ventilated with 100% O2 under similar conditions had an a/A ratio of 0.58 ± 0.06 after 24 h; data not shown). Two lambs with PPHN treated with 100% O2 alone died during the study at approximately 10 and 16 h after birth with severe hypoxemia, acidosis, and hypotension (Figure 1). Ventilation with 20 ppm iNO continuously from birth improved oxygenation, and the a/A ratio at 24 h was significantly increased in these lambs (0.51 ± 0.1; Figures 1 and and2).2). A single dose of intratracheal rhSOD at the time of delivery or at 4 h of age resulted in similar improvements: a/A ratios of these lambs were 0.48 ± 0.12 and 0.55 ± 0.09 at 24 h, respectively, significantly better than the oxygen-only group, and similar to the iNO group (Figure 1). Lambs that received rhSOD at birth in combination with iNO also had significantly improved oxygenation, with an a/A ratio of 0.46 ± 0.08 at 24 h. Oxygenation improved more rapidly with the combination of rhSOD and iNO compared with either intervention alone (a/A ratio > 0.3 by 2 h vs. 8–10 h; Figure 2; p < 0.05). The a/A ratios in the rhSOD + iNO group were significantly higher at 2, 6, and 8 h compared with lambs that received iNO only (Figure 2). However, there were no significant differences in a/A ratios by 24 h among any treatment groups (Figures 1 and and22).
The physiologic outcome with the final arterial blood gas results, mean airway pressure, and OI measurements are shown in Table 2. Although two lambs died in the 100% oxygen–alone group, all lambs receiving rhSOD and/or iNO survived. There were no significant differences in the pH, Pco2, bicarbonate, and base excess values among the five groups. However, PaO2 and OI values were significantly better in lambs that received rhSOD and/or iNO compared with lambs ventilated with 100% O2 alone. Lambs in the combined rhSOD + iNO group also had significantly lower mean airway pressures compared with those that received iNO alone at 24 h.
Cumulative NE concentration–response curves are shown in Figure 3. PAs isolated from newborn PPHN lambs studied with a normoxic gas mixture (20% O2, 6% CO2, and 74% N2) in the tissue bath showed a similar contraction response as those studied with a hyperoxic gas mixture (94% O2, 6% CO2). Therefore all PAs isolated from ventilated lambs were studied in 94% O2 baths.
Contractility in response to NE significantly increased in lambs ventilated with 100% oxygen compared with newborn PPHN lambs killed at birth. Ventilation with iNO significantly decreased this response as did intratracheal administration of rhSOD (at both time points). The contractile responses to 118 mM of KCl expressed as grams of force/grams of tissue weight followed a trend similar to NE (PPHN newborn, 150 ± 21; 100% oxygen, 225 ± 31 [p < 0.05 compared with newborn]; SOD at birth, 123 ± 12 [p < 0.05 compared with 100% oxygen], and SOD at 4 h, 155 ± 55; NO, 150 ± 21, and NO + SOD, 102 ± 18 [p < 0.05 compared with 100% oxygen]).
Lung 8-isoprostane F2α tissue concentrations were expressed as nanograms/milligrams lung protein and are shown in Figure 4. Ventilation with 100% oxygen dramatically increased 8-isoprostane tissue concentrations, an effect that was mitigated by pretreatment with either rhSOD or iNO (Figure 4). However, lung isoprostane tissue concentrations in the lambs that received both rhSOD and iNO did not significantly differ from the 100% O2–alone group (Figure 4).
Lung sections showing 3-nitrotyrosine fluorescent images (and comparative light microscopic images in the adjacent panel) are shown in Figure 5A. Fluorescent intensity of similarly sized PAs from these sections are shown as a fold of intensity of newborn PAs in Figure 5B. Ventilation with 100% O2 increased 3-nitrotyrosine fluorescence in PAs by approximately twofold compared with nonventilated newborn PAs. Ventilation with 100% O2 + iNO significantly increased 3-nitrotyrosine fluorescence by approximately ninefold. In sharp contrast, administration of a single dose of intratracheal rhSOD at birth and subsequent ventilation with 100% O2 + iNO significantly decreased 3-nitrotyrosine fluorescence in PAs (Figure 5).
SODs catalyze the conversion of superoxide anions to hydrogen peroxide (H2O2) and O2. Superoxide anions react with NO more efficiently than with any other known molecule and at a rate three times faster than dismutation by SOD (26, 27). Due to the efficiency of this reaction, the local concentration of SOD is a key determinant of the biological half-life of NO (6). Previous studies have established that intratracheal rhSOD is protective in newborn models of lung injury with hyperoxia and iNO (28), and its safety profile is well established in human neonates (12–14). Hence, SOD supplementation in infants with PPHN being managed with hyperoxic ventilation could be efficacious by (1) scavenging superoxide anions and (2) enhancing the bioactivity of both endogenous and exogenous NO and preventing peroxynitrite formation.
We found that a single intratracheal dose of rhSOD (administered at birth or at 4 h of age) in neonatal lambs with PPHN resulted in sustained increase in oxygenation over a 24-h period, similar to that found with iNO (Figure 1). The strategy behind delayed administration of rhSOD was to simulate the usual time frame needed to establish the diagnosis and initiate specific therapy for PPHN. Kinsella and colleagues previously demonstrated that both intratracheal rhSOD and iNO substantially improved oxygenation in extremely premature lambs, a finding similar to the current study (29). However, in contrast to our findings, delayed rhSOD treatment (at 2 h of age) did not result in significant improvement in preterm lambs (29). These contrasting findings suggest that the more immature lung may be more profoundly susceptible to the effects of hyperoxic mechanical ventilation.
Combined therapy with rhSOD and iNO resulted in a more rapid and sustained increase in oxygenation between 2 and 8 h of life compared with iNO alone (Figure 2). Moreover, the combination of rhSOD and iNO resulted in reduced ventilator pressure requirements compared with iNO alone (Table 2). However, the mean a/A ratios were not significantly different in the 10- to 24-h time period. It is possible that, in the presence of iNO, redosing of intratracheal rhSOD every 8 to 12 h may be necessary to offset free-radical damage induced by a combination of iNO and hyperoxia. Further studies are required to evaluate the efficacy of repeat doses of rhSOD in PPHN.
It is not clear what mechanisms are responsible for the physiologic improvements seen after rhSOD administration to lambs with PPHN. Traditionally, pulmonary hemodynamic measurements are done to assess the effect of various agents on PA pressure and pulmonary blood flow. However, the placement of these catheters and transducers requires a thoracotomy, which significantly contributes to the increased morbidity and mortality of these already critical lambs. Our previous short-term experiments indicated a significant decrease in PA pressure after intratracheal administration of rhSOD in a similar model of ovine PPHN (14), and we suspect that this results in enhanced ventilation–perfusion matching and decreased extrapulmonary right to left shunting. It is interesting that intravenous SOD and catalase did not attenuate or abolish group B streptococcus–induced pulmonary hypertension in 3- to 7-d-old lambs (30). Similarly, intravenous rhSOD did not alter arterial oxygenation (or cerebral microcirculation) in asphyxiated newborn piglets (31). We speculate that intratracheal administration of rhSOD, as opposed to the systemic circulation, is a more effective and selective pulmonary vasodilator. This beneficial effect could result from delivery directly to the site of hyperoxic injury (airway), improvement in ventilation–perfusion matching, and/or selective pulmonary vasodilation due to prolonging the bioavailability of NO in the lung.
It is not clear if rhSOD exerts these effects by acting within the alveoli or at the level of pulmonary vascular smooth muscle (either extra- or intracellular). Intratracheal administration of 5 mg/kg of rhSOD results in increased SOD activity in the lung, and this increase persists for at least 12 h (32). Despite its size (~ 32 kD) and negative polarity, rhSOD administered through the airway is taken up rapidly by airway epithelium (32). In preterm infants, intratracheal rhSOD was absorbed and excreted in active intact form in the urine (16). In addition, we administered rhSOD with surfactant. Surfactant has been shown to increase SOD activity in the bronchoalveolar lavage fluid and lung homogenates at 1 and 24 h after administration (33) and also prolong the half-life of rhSOD in lung tissue (34). We also administered surfactant as a vehicle (placebo) to the PPHN lambs ventilated with 100% O2 and with oxygen and iNO (100% O2 + NO group). It has been speculated that rhSOD may enter the cell either directly or indirectly, by combining with surfactant and forming liposomes that facilitate entry into the cell (32).
We conducted isolated vessel experiments to study the effect of oxygen ventilation with and without iNO and rhSOD on PA contractile response to a receptor-dependent α-adrenergic agent (NE) and receptor-independent agent (KCl). We and others have reported that PA contractility to NE decreases after birth in lambs (35) and piglets (36). However, ventilation with 100% O2 resulted in a substantial increase in contractile response to NE (Figure 3) and KCl. We speculate that the increased contractile response in lambs exposed to 100% O2 is secondary to the formation of ROS and their metabolites in the PAs. It has been recently reported that hyperoxic ventilation of isolated, perfused rat lungs can result in increased ROS and increased calcium in lung capillary endothelial cells (37). The role of superoxide anions in increasing pulmonary arterial contractility has also been reported (38, 39). Excess superoxide may react directly with NO, disrupting its physiologic signaling and forming peroxynitrite (6). Peroxynitrite has been shown to be a potent vasoconstrictor of newborn rat pulmonary arteries (11), possibly mediated by formation of isoprostanes (40). Newborn PAs exposed to peroxynitrite showed a 10-fold increase in isoprostane levels (11). Elevated tracheal aspirate isoprostane levels have been reported in babies ventilated with high levels of oxygen as compared with those ventilated with room air (41).
Neonatal PAs appear to be particularly sensitive to constrictor effects of isoprostanes. Isoprostanes generated more contraction force in newborn rats compared with adults exposed to oxygen (42). Recently, Gonzalez-Luis and colleagues reported that PAs from neonatal (12–24 h old) piglets are more sensitive to the contractile effect of isoprostanes compared with 2-wk-old piglets (43). We measured lung 8-isoprostane F2α tissue concentrations and found that ventilation with 100% oxygen dramatically increased these tissue concentrations, an effect that was reversed by rhSOD treatment. A similar decrease in lung 8-isoprostane tissue concentrations after treatment with SOD and catalase has been previously reported in infant rats and isolated ferret lungs (44, 45). We speculate that hyperoxic ventilation results in an ROS-induced increase in isoprostane tissue concentrations, producing increased pulmonary arterial contractility.
A significant increase in serum nitrotyrosine residues has been described in neonates treated with 24 h of iNO (46). Similarly, we found that ventilation with 100% O2 + iNO significantly increased 3-nitrotyrosine staining in PAs. Administration of intratracheal rhSOD before exposure to iNO prevented this increase in 3-nitrotyrosine staining. Oishi and colleagues have reported similar changes in 3-nitrotyrosine staining with iNO and polyethylene glycol–SOD administered through the PA catheter in 1-mo-old lambs (47). An increase in 3-nitrotyrosine staining in lambs receiving iNO reflects increased peroxynitrite formation. A potential benefit of supplementation with rhSOD appears to be a decrease in the formation of this toxic compound in the lungs after iNO therapy.
There are significant limitations to the current study. The benefit of rhSOD may be limited to infants with vascular remodeling–associated idiopathic or primary PPHN, similar to the ductal ligation model. Its benefit in PPHN secondary to parenchymal diseases of the lung, such as pneumonia, meconium aspiration, and hyaline membrane disease, needs to be further studied. Second, the lambs evaluated in this study were at 134 to 135 d gestation and were delivered by cesarean section. These factors (near-term gestation and lack of labor) may contribute to poor lung compliance. However, all lambs in our study received surfactant. Delaying ductal ligation and delivery at a later gestation has resulted in increased rates of spontaneous delivery and death of PPHN lambs in our laboratory. We did not wean oxygen concentrations in lambs with high PaO2 as would normally occur in the clinical management of babies with PPHN. By design, all lambs were exposed to approximately 100% O2 for the 24-h period so that measurements of oxygenation, isolated vessel responses, and tissue concentrations of isoprostanes and 3-nitrotyrosine could be easily compared between the groups. We acknowledge that continued exposure to high oxygen concentrations could have influenced our results. Last, we did not conduct any direct experiments linking high isoprostane levels to the increased contractile response to NE and KCl, and our findings could reflect an association of two unrelated results of ROS-mediated oxidant injury.
We conclude that rhSOD administered intratracheally, in a dose whose safety profile has been established in clinical trials (16, 17, 48, 49), significantly improves oxygenation, reduces ventilatory requirements, and reduces pulmonary arterial contractility and lung 8-isoprostane F2α levels in neonatal lambs with PPHN. The effect of a single dose of rhSOD administered either at birth or at 4 h of age is similar to iNO, at 20 ppm. We propose that rhSOD will be a cost-effective drug that can improve oxygenation early in the course of PPHN before significant hyperoxic insult occurs.
The authors thank Edmund A. Egan, M.D., ONY Laboratories, for providing calfactant (Infasurf) and INO Therapeutics for providing iNO and INOvent. They also thank Frederick C. Morin III, M.D., and Daniel D. Swartz, Ph.D., for their advice, and Bobby Mathew, M.R.C.P., Karen Wynn, R.N., N.N.P., Huamei Wang, M.D., Hschi-chi Koo, Ta Shauna Goldsby, Jan Capell, R.N., Margaret Brick, and Sharon Baumgartner for their expert technical assistance.
Supported by grant HL-54705 (R.H.S.) from the National Institutes of Health.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200605-676OC on September 28, 2006
Conflict of Interest Statement: S.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.A.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.W. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. S.F.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.A.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.M.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.H.S. received $2,500 per year from 2003–present for serving on an advisory board for INO Therapeutics.