1Pediatric Heart Lung Center, University of Colorado School of Medicine, Denver, Colorado; and 2Faculté de Médecine, Université de Lille II, Lille, France
Submitted 16 March 2005 ; accepted in final form 14 June 2005
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ABSTRACT |
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pulmonary hypertension of the newborn; lung; vasodilator; pulmonary vascular reactivity; direct soluble guanylate cyclase activator
The nitric oxide (NO)-cyclic 3'-5'-guanosine monophosphate (cGMP) cascade plays a major physiological role in the fetal and neonatal pulmonary circulation. NO is produced during conversion of L-arginine to L-citrulline by NO synthase (NOS) in endothelial cells and activates soluble guanylate cyclase (sGC) in vascular smooth-muscle cells to release cGMP. Past studies have shown that sGC is present and active early in the fetal lung (2, 16). Basal and stimulated NO release modulates pulmonary vasoregulation during late gestation. NOS antagonism increases PVR in near-term fetal lambs (1, 29). In addition, NOS inhibition selectively attenuates the pulmonary vascular response to acetylcholine, oxygen, shear stress, and myogenic response (1, 8, 22, 39). At birth, pretreatment with N-nitro-L-arginine, an NOS antagonist, reduces the fall in PVR and compromises the transition to neonatal circulation (1). Mechanisms that cause PPHN are uncertain, but previous studies suggest that impaired endothelial function with decreased NO production may contribute to PPHN (33, 41, 42).
Inhaled NO (iNO) is an effective therapy that improves gas exchange in neonates with severe pulmonary hypertension (7, 28, 31). However, past multicenter trials demonstrate that up to 40% of patients do not respond fully to iNO and require extracorporeal membrane oxygenation due to persistently elevated PVR and hypoxemia (7, 28, 31). These findings suggest that novel therapeutic strategies to stimulate pulmonary vasodilation or augment the response to iNO are still required to further improve outcomes in newborns with severe PPHN.
Direct pharmacological activators of sGC, such as BAY 41-2272, have been recently developed (36). BAY 41-2272 directly stimulates sGC on an NO-independent but heme-dependent site (36). Recent studies have shown that BAY 41-2272 causes potent vasodilation in the adult systemic and lung circulation (6, 10, 36, 37). Recently, BAY 41-2272 infusion has also been shown to cause potent and sustained pulmonary vasodilation in the fetus independently of endogenous NO release (9). Whether BAY 41-2272 is a potent vasodilator in the setting of chronic pulmonary hypertension has not been previously studied. In addition, the ability of BAY 41-2272 to augment the pulmonary vasodilation in responses to iNO in experimental PPHN is unknown.
Therefore, we hypothesized that therapeutic agents that cause vasodilation by directly stimulating sGC, such as BAY 41-2272, may provide an effective treatment of PPHN. To test this hypothesis, we studied the pulmonary vascular effects of BAY 41-2272 during the development of progressive pulmonary hypertension after partial ligation of the DA in chronically prepared, late-gestation fetal sheep. We report that BAY 41-2272 causes potent pulmonary vasodilation and improves oxygenation at birth in sheep with experimental PPHN and that BAY 41-2272 augments the response to iNO.
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METHODS |
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All procedures were reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center (Denver, CO). Surgery was performed between 125 and 130 days of gestation, according to previously published methods (4). Twelve mixed-breed (Columbia-Rambouillet) pregnant ewes were fasted for 48 h before surgery. Ewes were sedated with intramuscular buprenex (0.6 mg) and intravenous ketamine (60 mg) and diazepam (10 mg) and intratracheally intubated. Ewes were anesthetized with inhaled isoflurane (23%) and remained sedated but breathed spontaneously throughout surgery. Under sterile conditions, the left forelimb of the fetal lamb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with 1% lidocaine. Polyvinyl catheters were inserted into the axillary artery and advanced into the ascending aorta (Ao) and the superior vena cava. A left axillary to sternal thoracotomy exposed the heart and great arteries. Polyvinyl catheters were inserted into the left pulmonary artery (LPA), the main pulmonary artery (MPA), and left atrium (LA) by direct puncture and secured into position with purse-string sutures, as previously described. A 6-mm ultrasonic flow transducer (Transonic Systems, Ithaca, NY) was placed around the LPA to measure blood flow to the left lung [LPA blood flow (QLPA)]. The DA was exposed by blunt dissection. A cotton umbilical tape was placed around the DA and tightened around a right-angle surgical instrument to partially constrict the DA in a uniform manner. A catheter was placed in the amniotic cavity to serve as a pressure referent. The thoracotomy incision was closed in layers. The uteroplacental circulation was kept intact, and the fetus was gently replaced in the uterus. Ampicillin (500 mg) was added to the amniotic cavity before closure of the hysterotomy. Postoperatively, ewes were allowed to eat and drink ad libitum. All catheters were gently infused daily with 12 ml of normal (0.9%) saline with added heparin to maintain catheter patency.
Physiological Measurements
The Ao, MPA, and LA catheters were connected to a computer-monitored pressure transducer and recorder (Biopac Systems, Santa Barbara, CA). Pressures were referenced to amniotic pressure, and the pressure transducer was calibrated with a mercury manometer. The flow transducer cable was attached to an internally calibrated flow meter (Transonic Systems) for continuous measurements of QLPA. The absolute values of flow were determined from phasic blood flow signals as previously described (20). PVR in the left lung was calculated with the following equation: PVR (mmHg·ml1·min1) = (mean MPAP mean LAP)/QLPA, where MPAP is mean pulmonary artery pressure and LAP is left atrial pressure. Arterial blood gas tensions, pH, hemoglobin, oxygen saturation, and methemoglobin were measured from blood samples that were drawn from the Ao catheter and measured at 39.5°C with a blood gas analyzer and hemoximeter (model OSM-3; Radiometer, Copenhagen, Denmark).
Study Drugs
Acetylcholine (A-6625; Sigma-Aldrich, St. Louis, MO) was dissolved in saline to achieve a concentration of 15 µg/ml immediately before use. BAY 41-2272 (kindly provided by Dr. Johannes-Peter Stasch, Bayer, Pharma Research, Wuppertal, Germany) was dissolved with 50% ethanol (1:1 vol/vol ethanol-saline) and diluted with saline to achieve a concentration of 500 µg/ml. Sildenafil (1 mg/ml, intravenous solution; Pfizer, Sandwich, UK) was diluted with normal saline for a final concentration of 100 µg/ml.
Experimental Design
Protocol 1: acute pulmonary hemodynamic effects of BAY 41-2272, acetylcholine, and sildenafil during the development of chronic pulmonary hypertension. The purpose of this protocol was to compare the effects of acute pulmonary administration of acetylcholine, an endothelium-dependent vasodilator, BAY 41-2272, a direct activator of sGC, and sildenafil, a selective inhibitor of cGMP-specific phosphodiesterase type 5 (PDE5), during the development of chronic PH. The studies were performed 1 and 5 days after partial ligation of the DA. Saline (0.1 ml/min) was first infused into the LPA catheter for at least 30 min, and baseline hemodynamic measurements were recorded every 10 min for QLPA, MPAP, aortic pressure (AoP), LAP, and heart rate (HR). After baseline measurements were stable for a 30-min period, acetylcholine (15 µg/ml), BAY 41-2272 (500 µg/ml), and sildenafil (100 µg/ml) were infused at 0.1 ml/min in random order into the LPA for 10 min. The dose of acetylcholine used in this study was based on past studies that demonstrate a doubling of blood flow without systemic effects. The BAY 41-2272 and sildenafil doses were selected from previous studies that demonstrated comparable twofold increase in pulmonary blood flow in fetal sheep (9). After each infusion, the catheter was subsequently flushed with saline (0.1 ml/min). Hemodynamic measurements were recorded for at least 30 min after the return to baseline values before the next drug infusion. Arterial blood gas tensions were obtained before and after each study period.
Protocol 2: independent and combined effects of iNO, BAY 41-2272, and sildenafil in newborn sheep with chronic pulmonary hypertension after birth. The purpose of this protocol was to compare the hemodynamic effects of BAY 41-2272 and sildenafil on the transitional pulmonary vasodilation and to determine whether the drugs would alter the pulmonary vasodilator response to iNO in experimental PPHN. Nine days after the initial surgery, cesarean-section deliveries were performed under inhalational isoflurane anesthesia to the ewe. The uterus was partially delivered through an abdominal incision, and the flow transducer cables and catheters were carefully freed from the maternal flank. After the injection of pancuronium bromide (0.1 mg/kg, inferior vena cava) to the fetus, a hysterotomy was performed, and the fetus was rapidly intubated. The fetus was extracted from the uterus, dried, and warmed. The animal was placed on a heating pad and ventilated with a time-cycled, pressure-limited mechanical ventilator (Infant Star 950; Infrasonics, San Diego, CA) with room air. Initial ventilator settings included rate of 30 breaths/min, a peak inspiratory pressure (PIP) of 35 cmH2O, a positive end-expiratory pressure of 6 cmH2O, and an inspiratory time (IT) of 1.0 s. Subsequent ventilator adjustments were made based on arterial blood gas values and chest wall excursion. Target blood gas parameters included achieving blood pH between 7.35 and 7.45, and arterial partial CO2 pressure (PaCO2) of 3545 Torr. If PaCO2 fell <35 Torr, PIP was carefully decreased to a minimum of 22 cmH2O. Ventilator rate and IT were gradually decreased thereafter if PaCO2 remained below the target value after reaching a PIP of 22 cmH2O. Alternatively, if PaCO2 increased >45 Torr, ventilator rate was increased by 5 breaths/min, and IT was decreased to maintain the inspiratory-to-expiratory ratio at 1:1. Hypotension, defined as mean arterial pressure <30 mmHg, was treated with a rapid infusion of normal (0.9%) saline (10 ml/kg over 5 min). Lambs were kept on a heating pad throughout the study. Saline (0.1 ml/min) was first infused into the LPA catheter for at least 30 min, and baseline hemodynamic measurements were recorded every 10 min (baseline period 1). These measurements included QLPA, MPAP, AoP, LAP, and HR. After baseline measurements were stable for a 30-min period, iNO (20 ppm) was delivered for 10 min. NO was stopped, and hemodynamic measurements were recorded until the return of these values to baseline. After a 30-min baseline period (baseline period 2), sildenafil (100 µg/ml) or BAY 41-2272 (500 µg/ml) was infused in random order at 0.1 ml/min into the LPA for 10 min. After sildenafil or BAY 41-2272 infusion was stopped, hemodynamic measurements were recorded for at least 30 min after the return to baseline values before the next drug infusion (baseline period 3). The same protocol was repeated with sildenafil and BAY 41-2272 combined with iNO (20 ppm) for 10 min. Arterial blood gas tensions were obtained before and after each study period. At the end of the delivery study, sheep were killed with a large dose of pentobarbital sodium. Body weight was recorded, and the heart and lungs were rapidly removed in bloc through a midline thoracotomy. To assess the development of RVH after 9 days of DA compression, we weighed the free wall of the right ventricle and the left ventricle plus septum separately. RVH was expressed as the proportion of weights of the right ventricle and the left ventricle plus septum [RVH = RV/(LV + S)].
Statistical Analysis
Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (SAS Institute, Cary, NC). Statistical comparisons were made by factorial and repeated-measures analysis of variance and Fisher's protected least-significant-differences test for post hoc comparison. P < 0.05 was considered significant. In each experiment, n represents the number of different animals studied.
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RESULTS |
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Hemodynamic effects of partial constriction of DA. Chronic partial constriction of DA increased mean PAP by 22% (from 57 ± 3 at day 1 to 70 ± 4 mmHg at day 5, P < 0.02, Fig. 1) and mean PVR by 32% (from 0.86 ± 0.07 at day 1 to 1.13 ± 0.15 mmHg·ml1·min1 at day 5, P < 0.05, Fig. 1). QLPA did not change during the study period (65 ± 3 at day 1 and 62 ± 5 ml/min at day 5).
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Treatment with iNO (20 ppm), sildenafil (10 µg/ml), and BAY 41-2272 (50 µg/ml) alone caused significant pulmonary vasodilation after cesarean-section delivery of hypertensive sheep, as reflected by changes in PVR, QLPA, and MPAP (P < 0.01 compared with baseline, Figs. 5 and 6 and Table 3).
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Sildenafil dramatically decreased PVR (Fig. 6) and improved oxygenation with a PaO2 of 93.4 ± 9.1 Torr (P < 0.05, Table 3). Sildenafil combined with iNO decreased PVR to a greater extent than did iNO alone (PVR, 0.61 ± 0.06 mmHg·ml1·min1 for iNO and 0.37 ± 0.10 mmHg·ml1·min1 for sildenafil, P < 0.02, Fig. 6). PaO2 increased to 100.4 ± 13.2 Torr during combined iNO and sildenafil therapy (P < 0.05, Table 3).
iNO did not have systemic effects (Table 3). Sildenafil and sildenafil combined with iNO reduced AoP by 7 and 8%, respectively (NS, Table 3). BAY 41-2272 and BAY 41-2272 combined with iNO reduced AoP by 9 and 11%, respectively (NS, Table 3). Arterial PCO2, pH, hemoglobin, and HR did not change during infusion of the drugs.
Mean lamb weight at the time of study was 3,704 ± 376 g, and the ratio of the RV to LV + S was 0.69 ± 0.05 (n = 5).
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DISCUSSION |
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In this study, we explored three major targets of the NO-cGMP cascade (Fig. 7). As illustrated, NO mediates vasodilatation by stimulating sGC in vascular smooth muscle cells. Enzyme activation by the binding of NO converts guanosine triphosphate to cGMP, which modulates the activity of cGMP-dependent kinases, cGMP-regulated phosphodiesterases, and cGMP-regulated ion channels, which regulate vasodilation (11). cGMP signaling is downregulated by PDE5 activity, which lowers intracellular cGMP content through the degradation of cGMP to 5'-GMP (11). As in many settings, acetylcholine stimulates endothelial NOS (eNOS) and increases endothelial NO release in fetal sheep (1). BAY 41-2272 directly stimulates sGC at a NO-independent but heme-dependent site (36). Sildenafil inhibits PDE5 and enhances NO-induced vasorelaxation by increasing vascular smooth muscle cGMP concentration (11).
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In our study, chronic pulmonary hypertension was induced by partial compression of the DA in late-gestation fetal sheep. As previously described, DA ligation increases MPAP and PVR without causing sustained elevation of pulmonary blood flow or hypoxemia (22, 23, 38). In addition, chronic intrauterine pulmonary hypertension alters vasoreactivity, as reflected by downregulation of eNOS expression and altered endothelium-dependent vasodilation (22, 23, 33, 42). Over time, intrauterine pulmonary hypertension induces RVH and pulmonary vascular remodeling with increased smooth muscle cell hyperplasia, as observed in fatal human PPHN (3).
Multiple abnormalities in the NO-cGMP cascade contribute to mechanisms underlying endothelial dysfunction associated with chronic intrauterine pulmonary hypertension. Past studies have shown that chronic pulmonary hypertension decreases lung eNOS mRNA and protein expression and total NOS activity (42). eNOS activity is further impaired by altered heat shock protein 90-eNOS interaction (18), and increased superoxide generation may further limit NO bioactivity (34). In addition, in vitro studies demonstrated that sGC activity is impaired, resulting in a decreased generation of cGMP and reduced vascular relaxation to NO stimulation (38). Several studies have suggested that an increase in PDE5 activity could contribute to the pathophysiology of pulmonary hypertension. PDE5 activity is markedly elevated in fetal lamb with partial chronic DA ligation, suggesting that rapid cGMP hydrolysis may limit cGMP-dependent pulmonary vasodilation (12, 13). Thus chronic intrauterine pulmonary hypertension disrupts NO-cGMP signaling by decreasing eNOS expression and activity, altering sGC content and activity, and increasing PDE5 activity. Each pathway suggests potential alternate strategies to counteract the underlying pathophysiology of PPHN by treatment with exogenous NO, sGC activators, and PDE5 inhibitors.
In this model, BAY 41-2272 and sildenafil, but not acetylcholine, caused pulmonary vasodilation despite progressive increase in PVR. Interestingly, BAY 41-2272-induced pulmonary vasodilation was even greater at day 5 than at day 1. The mechanism of this enhanced effect is unclear. Mullershausen et al. (26) found that, in addition to direct stimulation of sGC, BAY 41-2272 may have some PDE5 inhibitor effects. In contrast, Stasch et al. (36) reported that BAY 41-2272 is an NO-independent sGC activator without any PDE5 inhibitory activity. Our previous study in the normal fetal sheep demonstrated that BAY 41-2272-induced pulmonary vasodilation was not blocked by nitro-L-arginine and the vasodilator effects of BAY 41-2272 were more sustained than those observed during treatment with sildenafil (9). Whether BAY 41-2272 at high doses can inhibit other phosphodiesterase isoforms is uncertain in our study (21). However, a role of additional phosphodiesterase isoforms in PPHN is currently unexplored and further studies are needed to fully examine the mechanisms responsible for this response. In addition, studies in pulmonary arteries isolated from fetal lambs with pulmonary hypertension demonstrated impaired relaxations to agents that stimulate endothelial NO production and cGMP production by sGC but normal relaxation to cGMP given exogenously or produced by particulate guanylate cyclase (38). Despite downregulation of lung sGC content and activity (38), our findings demonstrate persistent and potent pulmonary vasodilation by BAY 41-2272. We speculate that this effect may be partly due to its unique ability to activate sGC (36).
Several studies in perinatal animals and in human newborns demonstrated iNO as a potent and selective pulmonary vasodilator in transitional pulmonary circulation at birth, especially in the settings of impaired NO production, such as PPHN (7, 17, 28, 30, 31). However, some newborns have poor or partial response to iNO therapy. Mechanisms that contribute to this problem include poor lung inflation during mechanical ventilation, impaired cardiac function, and anatomic lung disease. In some cases, enhancement of the vascular response to iNO may be achieved via augmentation of the NO-cGMP cascade. Sildenafil causes pulmonary vasodilation in newborns and adults and has been proposed for primary treatment of pulmonary hypertension (32, 35, 40, 43). In animal models of acute pulmonary hypertension, intravenous sildenafil induces a potent pulmonary vasodilator effect (14, 35, 43) and nebulized sildenafil augments the iNO-induced pulmonary vasodilation (14). In our study, BAY 41-2272 was at least as effective as iNO or sildenafil to reduce PVR in newborn sheep with chronic pulmonary hypertension. In addition, BAY 41-2272 augmented the pulmonary vasodilator effect to iNO. This data suggest that BAY 41-2272 may sensitize sGC to become more responsive to NO, as suggested by its effects on platelets (26).
In this study, BAY 41-2272 and sildenafil did not decrease systemic arterial pressure; however, these agents were infused directly into the LPA. We suspect that these drugs may reduce systemic pressure if administered into the systemic circulation, as would likely occur in the clinical setting. Furthermore, this study examined only the acute effects of BAY 41-2272 and sildenafil in this model of PPHN. The vasodilator effect of BAY 41-2272 was sustained during prolonged infusions in the normal fetus (9), but whether continuous BAY 41-2272 infusion can sustain its effects in sheep with experimental PPHN remains unknown.
In conclusion, BAY 41-2272 causes potent pulmonary vasodilation in fetal sheep during the progressive increase of pulmonary hypertension in utero. Moreover, BAY 41-2272 causes selective and potent pulmonary vasodilation and augments the pulmonary vasodilator response to iNO during transition at birth in sheep with chronic pulmonary hypertension. These observations suggest the therapeutic potential of BAY 41-2272 as an alternate or adjuvant therapy for severe neonatal pulmonary hypertension, leading to our speculation that BAY 41-2272 could provide a novel treatment or strategy for severe PPHN.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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