Sections of 1 Cardiology, 2 Neonatology, and 3 Pulmonary Medicine, Pediatric Heart Lung Center, University of Colorado School of Medicine, and The Children's Hospital, Denver, Colorado 80218
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ABSTRACT |
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Endothelin (ET)-1 contributes to regulation of pulmonary vascular tone and structure in the normal ovine fetus and in models of perinatal pulmonary hypertension. The hemodynamic effects of ET-1 are due to activation of its receptors. The ETA receptor mediates vasoconstriction and smooth muscle cell proliferation, whereas the ETB receptor mediates vasodilation. In a lamb model of chronic intrauterine pulmonary hypertension, ETB receptor activity and gene expression are decreased. To determine whether prolonged ETB receptor blockade causes pulmonary hypertension, we studied the hemodynamic effects of selective ETB receptor blockade with BQ-788. Animals were treated with an infusion of either BQ-788 or vehicle for 7 days. Prolonged BQ-788 treatment increased pulmonary arterial pressure and pulmonary vascular resistance (P < 0.05). The pulmonary vasodilator response to sarafotoxin 6c, a selective ETB receptor agonist, was attenuated after 7 days of BQ-788 treatment, demonstrating pharmacological blockade of the ETB receptor. Animals treated with BQ-788 had greater right ventricular hypertrophy and muscularization of small pulmonary arteries (P < 0.05). Lung ET-1 levels were threefold higher in the animals treated with BQ-788 (P < 0.05). We conclude that prolonged selective ETB receptor blockade causes severe pulmonary hypertension and pulmonary vascular remodeling in the late-gestation ovine fetus.
nitric oxide; persistent pulmonary hypertension of the newborn; congenital heart disease; pulmonary circulation; BQ-788
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INTRODUCTION |
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PULMONARY VASCULAR RESISTANCE (PVR) is elevated in the normal fetal lung, and pulmonary blood flow accounts for <8-10% of the combined ventricular output of blood from the heart (15). Mechanisms responsible for the maintenance of high PVR in the fetus include the lack of an air-liquid interface, low oxygen tension, decreased vasodilator activity, increased vasoconstrictor activity, and high myogenic tone (2, 6, 11, 37, 38). Endothelium-derived products, including vasodilator stimuli such as nitric oxide (NO) and prostacyclin, and vasoconstrictor stimuli such as leukotrienes and endothelin-1 (ET-1), contribute to vascular tone in the fetal lung (1, 6, 9-11, 18, 37, 41).
The endothelins (ETs) are a family of isopeptides with potent vasoactive properties. ET-1 is present in the perinatal lung (29) and is vasoactive in the fetus (8, 17, 18, 23, 43). The actions of ET-1 are dependent on activation of at least two receptor subtypes: ETA and ETB. ETA receptors are located on smooth muscle cells and mediate vasoconstriction and smooth muscle proliferation (16, 44). Stimulation of endothelial ETB receptors causes vasodilation through release of NO and also functions to remove ET-1 from the circulation (13, 14, 18, 36). Past studies have shown that ETB receptors on pulmonary vascular smooth muscle mediate vasoconstriction in some species (34); however, ETB receptors are present only on the endothelium in the ovine fetal lung (17, 18). Acute blockade of the ETB receptor does not change basal pulmonary tone in the ovine fetus (20, 22); however, prolonged blockade of the ETB receptor has not been studied. Prolonged blockade of the ETB receptor may lead to diminished NO or prostacyclin production, elevated ET-1 concentrations, or enhanced ETA receptor-mediated vasoconstriction. The physiological role of ET-1 in the regulation of vascular tone in the ovine fetal lung is complex, and the exact role of the ETB receptor in regulation of pulmonary tone remains incompletely understood.
Ligation of the ductus arteriosus in late-gestation fetal lambs has provided an experimental model for studying the mechanisms that contribute to the structural and functional changes associated with perinatal pulmonary hypertension (3, 5, 30, 31). Previous studies (19, 39) have demonstrated that ductus arteriosus ligation alters production of endothelium-derived products such as NO and ET-1. Chronic intrauterine pulmonary hypertension caused by ductus arteriosus ligation leads to abnormalities of the ET system, such as diminished ETB receptor-mediated vasodilation in combination with enhanced ETA receptor-mediated vasoconstriction (22). Furthermore, this model is characterized by decreased ETB receptor mRNA expression (19). However, it is not known whether deficiency of the ETB receptor is a marker or mediator of pulmonary hypertension in fetal lambs.
Based on these studies, we hypothesized that the ETB receptor plays an important role in the chronic regulation of pulmonary vascular tone and structure in the ovine fetus and that prolonged ETB receptor blockade would cause pulmonary hypertension. Although acute blockade of the ETB receptor does not change basal pulmonary tone in the fetal lung, chronic blockade of the ETB receptor may have different physiological effects due to the role of the ETB receptor in the production of NO or the clearance of ET-1. To test this hypothesis, we studied the effects of selective ETB receptor blockade with BQ-788 delivered by continuous infusion into the left pulmonary arteries (LPAs) of fetal lambs. The effects of prolonged ETB receptor blockade in utero were evaluated both in the fetuses and after cesarean section delivery. Hypertensive lung structural changes, right ventricular hypertrophy (RVH), plasma ET-1 levels, and lung endothelial nitric oxide synthase (eNOS) protein content were studied after 7 days of BQ-788 treatment.
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METHODS |
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Surgical preparation and physiological measurements. All procedures and protocols were previously reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Nine mixed-breed (Columbia-Rambouillet) pregnant ewes between 125 and 129 days gestation (term = 147 days) were fasted 24 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (2-4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Ewes were kept sedated with pentobarbital sodium but breathed spontaneously throughout the surgery. Penicillin (500 mg) and streptomycin (1 g) were administered to the ewes at surgery. Under sterile conditions, each fetal lamb's left forelimb was delivered through a uterine incision. A skin incision was made under the left forelimb after local infiltration with 2-3 ml of lidocaine (1% solution). A polyvinyl catheter was advanced into the ascending aorta (Ao) and the superior vena cava after insertion into the axillary artery and vein, respectively. A left axillary-to-sternal thoracotomy exposed the heart and great arteries. Catheters were inserted into the LPA, the main pulmonary artery (MPA), and the left atrium (LA) by direct puncture through purse-string sutures as previously described (24). Catheters were guided into position with a 14- or 16-gauge intravenous placement unit (Angiocath; Travenol, Deerfield, IL). These catheters were secured by tightening the purse-string suture as the introducer was withdrawn. The LPA catheter was inserted at the bifurcation of the MPA and the ductus arteriosus and guided through the common pulmonary artery into the LPA. The MPA catheter was inserted between the ductus arteriosus and the pulmonic valve. A 6.0-mm ultrasonic flow probe (Transonic, Ithaca, NY) was placed around the LPA to measure LPA blood flow. The thoracotomy incision was closed in layers. The uteroplacental circulation was kept intact, and the fetus was gently replaced in the uterus, with exposed surfaces bathed in warm towels. Ampicillin (500 mg) was added to the amniotic cavity before closure of the hysterotomy. Ampicillin (250 mg) was infused daily into the fetal vein and amniotic cavity during the first 3 days after surgery. The ewes were allowed to recover.
The flow transducer cables were attached to an internally calibrated flowmeter (Transonic) for continuous measurement of LPA flow. The absolute values of the flows were determined from phasic blood flow signals obtained during baseline periods as previously described (18, 21). A correction factor between end-diastolic flow and the internally calibrated zero point on the Transonic flowmeter was added to the mean flow on the Transonic flowmeter. The value obtained from this method correlated with previously determined measures of LPA flow in the late-gestation ovine fetal lung (27). Calculations of resistances are reported as left lung PVR [in mmHg · mlDrug preparation. BQ-788 (Alexis Biochemicals, San Diego, CA), a selective ETB receptor antagonist, was dissolved in DMSO. This solution was diluted in 24 ml of normal saline and directly infused into the MPA over 24 h at a rate of 1 ml/h. DMSO (0.1% in normal saline) was infused at the same rate into control animals. Sarafotoxin 6c (SFX-S6c; Alexis Biochemicals) was dissolved in normal saline and infused into the LPA at a rate of 0.1 µg/min for 10 min based on previous studies (22).
Protocol 1: Hemodynamic effects of prolonged BQ-788 treatment in late-gestation fetal lambs. To determine the hemodynamic effects of prolonged ETB receptor blockade, either 1 mg/day of BQ-788 (n = 5 lambs) or the 0.1% DMSO control solution (n = 4 lambs) was infused into the LPA by continuous infusion for 7 days. Infusions were begun 1 day after recovery from surgery. To determine the effectiveness of ETB receptor blockade, SFX-S6c, a selective ETB receptor agonist, was infused before and after BQ-788 treatment.
Protocol 2: Effects of prolonged BQ-788 treatment on the vasodilator response to birth-related stimuli and inhaled NO. To determine whether prolonged ETB receptor blockade attenuates vasodilation and the decline in PVR at birth, we studied the hemodynamic and blood gas effects of prolonged BQ-788 treatment after cesarean section delivery and mechanical ventilation of the lambs in protocol 1 (n = 5, BQ-788; n = 4, control). During the delivery study, pancuronium bromide (0.3 mg to the vena cava) was administered to the fetus, and the fetal head was delivered. A tracheotomy was performed, and a 5.0-mm-ID endotracheal tube was inserted. Mechanical ventilation with 10% O2-90% N2 was initiated. The uterine incision was carefully extended to allow visualization of the thorax. Initial ventilator settings included a rate of 30 breaths/min, a peak inspiratory pressure of 30 cmH2O, a positive end-expiratory pressure of 5 cmH2O, and an inspiratory time of 0.8 s. Peak inspiratory pressure was increased until chest wall motion could be observed with inspiration (a maximum of 35 cmH2O). Rate was varied as necessary to maintain PCO2 close to 40 mmHg. The umbilical cord was ligated. Animals were then ventilated sequentially with 100% O2 and 100% O2 with 20 parts/million inhaled NO. Measurements were made after 30 min of each intervention.
Protocol 3: Effects of prolonged BQ-788 on RVH. To determine the effect of prolonged ETB receptor blockade with BQ-788 on cardiac hypertrophy, weights of the right ventricle and left ventricle plus septum were measured (BQ-788, n = 5 hearts; control, n = 4 hearts). The ratio of right ventricle to left ventricle plus septum weights was calculated.
Protocol 4: Effect of prolonged ETB receptor blockade with BQ-788 on lung vascular histology. To determine the effect of prolonged ETB receptor blockade with BQ-788 on the wall thickness (WT) of small pulmonary arteries, we performed morphometric evaluation of the lungs from animals killed in protocol 2 (BQ-788, n = 5; control, n = 4). Methods for examining lung histology and performing morphometric analysis included vascular perfusion of the pulmonary artery and tracheal inflation with 1% paraformaldehyde (21). After fixation, thin longitudinal sections were cut, placed in paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin. Morphometric analysis was performed by a blinded observer with a Zeiss interactive digital analyzer system. Pulmonary arteries <100 µm were landmarked according to their associated airways (terminal bronchiole, respiratory bronchiole, and alveolar duct); measurements included the WT and vessel diameter of at least 10 consecutive pulmonary arteries per animal. The WT of each artery was expressed as a percentage of the vessel diameter by the formula (2 × medial WT/external diameter) × 100. Pulmonary arteries from the right and left lungs of control and BQ-788-treated animals were examined.
Protocol 5: Measurement of lung ET-1 peptide. We measured left lung ET-1 protein content in lambs after prolonged ETB receptor blockade and also in control lambs. Eight lung samples (BQ-788, n = 4; control, n = 4) were homogenized in 1 ml of 1 M acetic acid-0.1% Triton X-100 (Sigma, St. Louis, MO) and immediately boiled for 7 min as previously reported (28). C2 columns (Amersham, Arlington Heights, IL) were equilibrated with 2 ml of methanol followed by 2 ml of deionized water. Forty microliters of homogenate were applied to the column and washed with 5 ml of 0.1% trifluoroacetic acid. The sample was eluted with 2 ml of 80% methanol-0.1% trifluoroacetic acid, and the volume was reduced for 5 h (to near dryness) on a Speed-Vac concentrator (Savant, Farmingdale, NY). Samples were reconstituted to 250 µl, and 100 µl of sample and standard were applied to replicate wells of the ET-1 peptide ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. The antibody used in this assay cross-reacts with ET-2 (45%) and ET-3 (14%). Activity was quantified by determination of optical density units at 450 nm with a DynaTech MR 700 plate reader (Bio-Tek, Winooski, VT).
Protocol 6: Western blot analysis for lung eNOS. Western blot analysis was performed according to our previously published techniques (25) with a monoclonal antibody to eNOS (Transduction Laboratories, Lexington, KY). Briefly, lung tissue (BQ-788, n = 4; control, n = 4) was homogenized in 25 mM Tris · HCl, pH 7.4, containing 1 mM EDTA, 1 mM EGTA, 0.1% (vol/vol) 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, 2 mM leupeptin, and 1 mM pepstatin A on ice. The homogenate was centrifuged at 1,500 g at 4°C for 10 min to remove cell debris. SDS-PAGE was performed on 25-g aliquots of homogenate protein with a 7.5% (wt/vol) polyacrylamide gel. Proteins were transferred to nitrocellulose paper with an electrophoretic transfer cell. The blot was blocked in 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 2% (vol/vol) BSA, and 0.1% (vol/vol) Tween 20 overnight at 4°C and then incubated with the primary antibody for 1 h at room temperature. eNOS antibody was diluted 1:500 in blocking buffer. The blot was then washed six times, 5 min per wash, with 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.1% (vol/vol) Tris-buffered saline-Tween 20 (TBS-T) at room temperature. The blot was incubated for 1 h with anti-mouse IgG antibody coupled to horseradish peroxidase diluted in blocking buffer. The blot was then washed six times, 5 min per wash, with TBS-T at room temperature, and protein bands were detected by chemiluminescence and exposure to X-ray film. Western blot analysis with increasing amounts of lung protein from a control animal showed that there was a linear increase in eNOS protein signal and that the amount of protein (25 µg) used for comparison fell within the linear range of the Western blot analysis. Densitometry was performed with a scanner and NIH image software (National Institutes of Health, Bethesda, MD).
Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Statview 4.5 software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made with ANOVA for repeated measures and Fisher's protected least significant difference test for post hoc comparisons. P < 0.05 was considered significant.
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RESULTS |
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Protocol 1: Hemodynamic effects of prolonged BQ-788 treatment in
late-gestation fetal lambs.
Baseline hemodynamic measurements and arterial blood gas tensions
between control and BQ-788 treatment groups were not different (Table
1). After 7 days of BQ-788 treatment,
left lung PVR was greater in the animals treated with BQ-788 than in
control animals (Fig. 1). Mean
PAP and mean AoP were greater in animals treated with BQ-788 than in
their control counterparts, but LPA flow and arterial blood gas
tensions were not different. Vasodilation to the selective
ETB receptor agonist SFX-S6c was attenuated after 7 days of
BQ-788 treatment (Fig. 2). There was a
52 ± 12% fall in PVR to SFX-S6c before initiation of BQ-788,
whereas after BQ-788, there was no change in PVR, which suggests
blockade of the ETB receptor.
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Protocol 2: Effects of prolonged BQ-788 treatment on the
vasodilator response to birth-related stimuli and inhaled NO.
At delivery, animals treated with BQ-788 had higher PAP and
PVR at baseline than control lambs (Fig.
3, Table
2.) During sequential ventilation with
low O2 (10%), high O2 (100%),
and inhaled NO with 100% O2, PAP remained higher at each
intervention in the fetuses treated with BQ-788 than in the control
group (Table 2). PVR in the left lung remained higher in the BQ-788
treatment group during ventilation with low (10%) and high
O2 (100%) and after inhaled NO. Mean AoP at baseline was
also higher in the animals treated with BQ-788 but not during the
remainder of the study. There was no difference in arterial blood gas
tensions between the groups throughout the study. Ventilation with low O2 did not lower PAP in either the control or
BQ-788-treated animals compared with the fetal baseline; however, the
addition of 100% O2 lowered PAP in the control animals. NO
inhalation lowered PAP in both the control and BQ-788-treated animals
compared with the response to 100% O2 alone. In the
control group, PVR fell in response to ventilation, 100%
O2, and NO compared with the prior treatment (fetal,
ventilation, or 100%). The animals treated with BQ-788 did not show a
drop in PVR compared with the prior treatment.
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Protocol 3: Effects of prolonged BQ-788 on RVH.
Prolonged treatment with BQ-788 increased the weight of the right
ventricle (6.3 ± 0.5 vs. 8.0 ± 0.2 g, control vs.
BQ-788, P < 0.05) but not the left ventricle plus
septum. RVH, as assessed by the ratio of right ventricular weight to
left ventricular plus septum weight, was greater with BQ-788 treatment
(Fig. 4).
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Protocol 4: Effect of prolonged ETB receptor blockade
with BQ-788 on lung vascular histology.
BQ-788 treatment increased the WT of small pulmonary arteries (Fig.
5). In the BQ-788-treated lungs, the %WT
was higher in the pulmonary arteries of the left lungs of the treated
animals than in the right lungs of treated animals or in either lung of the control animals (Fig. 6).
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Protocol 5: Measurement of lung ET-1 peptide.
Left lung ET-1 protein content was threefold higher in the animals
treated with BQ-788 than in control animals (Fig.
7).
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Protocol 6: Western blot analysis for lung eNOS.
Whole left lung eNOS protein content was not different in
BQ-788-treated versus control animals (Fig.
8).
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DISCUSSION |
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Whereas acute blockade of the ETB receptor did not change basal pulmonary tone in the ovine fetus, prolonged blockade of the ETB receptor with BQ-788 caused pulmonary hypertension, RVH, and increased muscularization of small pulmonary arteries. In response to birth-related stimuli, including ventilation with low oxygen and inhaled NO, PVR remained higher in lambs that were treated with BQ-788. These findings support the hypothesis that ETB receptor activity does not contribute to basal PVR, but its function contributes to chronic regulation of pulmonary vascular tone in the ovine fetal lung. These findings suggest that diminished ETB receptor function leads to chronic pulmonary hypertension in the perinatal period.
Although past studies (7, 8, 18, 23, 29, 42, 43) have demonstrated that ET is present in the fetal lung and contributes to the regulation of vascular tone during late gestation, the physiological roles of ET and its regulation in the normal ovine fetal pulmonary circulation are complex and incompletely understood. In the ovine fetal lung, the ETA receptor is located on vascular smooth muscle and mediates vasoconstriction. Selective ETA receptor blockade with BQ-123 causes sustained pulmonary vasodilation in the normal ovine fetus (18). In contrast, ETB receptors are present only on the endothelium in the ovine fetal lung (17, 18). The present study highlights the different roles of the ETB receptor in acute and chronic regulation of pulmonary vascular tone. Acute blockade of the ETB receptor does not change basal pulmonary tone in the ovine fetal lung (20, 22); however, prolonged blockade of the ETB receptor causes severe pulmonary hypertension.
The exact mechanism of the increase in PAP and PVR after prolonged ETB receptor blockade remains uncertain. Because lung ET-1 protein content was increased threefold, it is possible that the clearance functions of the ETB receptor are important in chronic regulation of tone. It is less likely that a marked decrease in NO production contributes to the hemodynamic abnormalities seen with chronic ETB receptor blockade in the ovine fetal lung because lung eNOS protein content was not decreased; however, direct NO production was not measured. Conversely, a decrease in prostaglandin production or potassium channel activation may contribute to the changes seen; however, these were not studied. A recent study (33) has suggested the importance of the ETB receptor in regulation of endothelin-converting enzyme expression, the enzyme responsible for production of ET-1. In this study, ETB receptor stimulation by ET-1 decreased endothelin-converting enzyme expression, thus potentially limiting ET-1 production. Prolonged ETB receptor blockade may lead to an increase in ET-1 production. Some of the hemodynamic changes after prolonged ETB receptor blockade may be due to systemic effects of BQ-788 because systemic arterial pressure was elevated. A previous study (26) of systemic arterial hypertension caused by renal artery stenosis caused pulmonary hypertension. However, greater medial hypertrophy was seen in the left lungs of the BQ-788-treated animals compared with the right lungs of treated animals, suggesting a local effect. Furthermore, there was no difference in left ventricular hypertrophy between treated and control animals. It is interesting that prolonged ETB receptor blockade caused hypertensive lung structural changes, whereas in utero blockade of NO production did not change pulmonary vascular structure (12). The impact of prolonged ETB receptor blockade on other vasodilator systems remains incompletely understood. We speculate that the ETB receptor plays a crucial role in regulation of pulmonary vascular tone in the ovine fetus.
Many of the hemodynamic changes noted after prolonged ETB receptor blockade were similar to the changes caused by ligation of the ductus arteriosus in fetal lambs. Pulmonary hypertension caused by ductus arteriosus ligation is a model for the clinical syndrome of persistent pulmonary hypertension of the newborn (3, 5, 21, 31, 32). Chronic intrauterine pulmonary hypertension caused by ligation of the ductus arteriosus causes diminished ETB receptor-mediated vasodilation (22) and decreased ETB receptor mRNA expression (19). Both chronic intrauterine pulmonary hypertension due to this ligation and prolonged ETB blockade cause elevation of intrauterine PAP, RVH, and hypertensive lung structural changes, as well as abnormal pulmonary vasoreactivity and failure to achieve the normal decline in pulmonary resistance at birth. We speculate that some of the hemodynamic abnormalities in clinical persistent pulmonary hypertension of the newborn may be due to altered function of the ETB receptor.
ET-1 levels are increased in human disorders of pulmonary hypertension. Elevated immunoreactive ET-1 levels have been found in persistent pulmonary hypertension of the newborn (35), children with pulmonary hypertension associated with congenital heart disease and bronchopulmonary dysplasia (4), and children with congenital heart disease and increased pulmonary blood flow (24, 40). However, the role of ET receptor antagonists in the treatment of pulmonary hypertension remains incompletely understood. This study suggests that the potential deleterious effects of ETB receptor blockade in the treatment of pulmonary hypertensive disorders should be considered.
In summary, prolonged ETB receptor blockade in the ovine fetal lung causes pulmonary hypertension characterized by elevated intrauterine PAP and resistance, abnormal pulmonary vasoreactivity, RVH, hypertensive lung structural changes, and elevated lung ET-1 protein content. We conclude that the ETB receptor contributes to chronic regulation of pulmonary vascular tone. We speculate that loss of ETB receptor activity may predispose affected individuals to the development of severe pulmonary hypertension.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Heart, Lung, and Blood Institute Grants K08-HL-03823-01A (to D. D. Ivy) and HL-51744 and by a grant from the March of Dimes Birth Defects Foundation (to D. D. Ivy).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. D. Ivy, Dept. of Cardiology, Box B100, The Children's Hospital, 1056 E. 19th Ave., Denver, CO 80218 (E-mail: dunbar.ivy{at}UCHSC.edu).
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.
Received 3 February 2000; accepted in final form 24 April 2000.
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