1Anesthesiology, 2Pediatrics, and 3Surgery, and the 4Cardiovascular Research Institute, University of California, San Francisco, California 94143
Submitted 7 August 2003 ; accepted in final form 23 January 2004
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ABSTRACT |
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congenital heart disease
Multiple studies have shown that increased pulmonary blood flow and/or pressure affect endothelial cell functions within the alveolar space. Grigg et al. (22) found elevated levels of endothelin-1 (ET-1) in bronchoalveolar lavage in children with congenital heart disease with increased pulmonary blood flow. Ishikawa et al. (36) found elevated plasma ET-1 levels in children with ventricular septal defects who had both volume and pressure overload to the pulmonary circulation but not in children with atrial septal defects who had only volume overload. In cultured human vascular endothelial cells, shear stress stimulated the synthesis of mRNA for platelet-derived growth factors-A and -B (48), tissue plasminogen activator (20), and intercellular adhesion molecule-1 (46).
Recently, increased pulmonary blood flow and/or pressure has also been shown to affect pulmonary epithelial cell functions. Wang et al. (59) showed that, in an isolated, constantly inflated, blood-perfused rat lung, increased left atrial pressure increased septal capillary diameter, which resulted in the exocytosis of lamellar bodies, hence surfactant secretion, in type II alveolar cells. In a model of stretch, Gutierrez et al. (23, 24) found that mechanical distention decreased the transcription of surfactant protein (SP)-B and SP-C in cultured rat alveolar type II cells.
In children with congenital heart disease with increased pulmonary blood flow, the changes in SP expression may play a significant role in the aberration in lung compliance (15, 16) and increased susceptibility to pulmonary infection (18) seen in this pathological state. The function of surfactant, a complex lipoprotein assembled and secreted in the alveolar space by type II cells, is to lower surface tension at the air-water interface of the lung alveoli, thereby stabilizing lung volume at low transpulmonary pressures. For example, premature neonates with respiratory distress syndrome, marked by a deficiency of surfactant at birth, when treated with exogenous surfactant have improved oxygenation, improved arterial-to-alveolar oxygen gradient, increased functional residual capacity, and increased lung compliance (21, 32). In addition, among neonates and infants with respiratory distress syndrome at risk of developing bronchopulmonary dysplasia, the composition and kinetic of surfactant phospholipids (PL) may change during the progression of the disease (11, 17, 37, 54). SP-A and SP-D are also lung collectins, which are important to innate immune response to viral, fungal, and bacterial challenges, as well as to inflammatory regulation within the lung. In vitro, SP-A binding to alveolar macrophages stimulates chemotaxis and binding to bacteria and viruses (18). Mice lacking SP-A or SP-D showed deficient uptake of bacteria by alveolar macrophages (4042). In premature baboons in a model of chronic lung disease, a decrease in SP-A and SP-D levels in bronchoalveolar lavages was associated with an increased risk of infection (2). Consequently, SP deficiency may result in eventual respiratory failure from decreased lung compliance and increase susceptibility to infections.
Previously, in an in vivo lamb model of congenital heart disease with increased pulmonary blood flow at 4 wk of age (52), we found a decrease in SP-A gene expression, as well as a decrease in SP-A and SP-B protein contents (25) associated with an increase in endothelial nitric oxide (NO) and ET-1 mRNA and protein levels (8, 9). Our present study utilized our lamb model of congenital heart disease with pulmonary hypertension to determine the effect of increased pulmonary blood flow on the expression of SP-A, SP-B, and SP-C within the first week of life. By characterizing the gradual changes in surfactant gene expression and protein content associated with increased pulmonary blood flow, we may better understand the development of pulmonary vascular resistance in relation to other vasoactive substances, such as NO and ET-1, and better anticipate the optimal timing of surgical intervention.
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METHODS |
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Ewes. Eight pregnant, mixed-breed Western ewes (135140 days gestation; term = 145 days) were operated on under sterile conditions. Through a left lateral fetal thoracotomy, an 8.0-mm Gore-Tex vascular graft (2-mm length; W. L. Gore, Milpitas, CA) was anastomosed between the ascending aorta and main pulmonary artery, as previously described in shunted lambs (52). After the surgery, the ewes were returned to the cage with free access to food and water. One gram of cefazolin and 100 mg of gentamicin sulfate were administered to the ewes intraoperatively and daily thereafter until 2 days following spontaneous delivery of the lambs.
Lambs.
After spontaneous delivery, the lambs were administered daily furosemide (1 mg/kg im) and one dose of elemental iron (50 mg im). Weight, respiratory rate, and heart rate were monitored daily. Within the first week of life, 16 lambs (8 shunted and 8 case-matched controls) were sedated with ketamine hydrochloride (15 mg/kg im), and, under local anesthetic, polyvinyl catheters were inserted into the artery and vein of one hind leg and advanced into the descending aorta and inferior vena cava, respectively. The lambs were then induced with ketamine hydrochloride (0.3 mg·kg1·min1), diazepam (0.002 mg·kg1·min1), and fentanyl citrate (1.0 µg·kg1·h1), intubated with an endotracheal tube (5.0- to 6.0-mm outer diameter), and mechanically ventilated with a Healthdyne (Marietta, GA) pediatric time-cycled, pressure-limited ventilator. An intravenous infusion of Lactated Ringer solution with 5% dextrose was started at 75 ml/h and continued throughout the study period. Succinylcholine chloride (2 mg/kg per dose) was intermittently given for muscle relaxation. Heart rate and systemic blood pressure were monitored continuously to ensure adequate anesthesia. Ventilation with a respiratory rate of 20 breaths/min, inhale-to-exhale ratio of 0.7, inspired O2 fraction of 21%, mean peak inspiratory pressure of 21 cmH2O, and an end-expiratory pressure of 5 cmH2O were adjusted to maintain an arterial PCO2 between 35 and 45 Torr and arterial PO2 between 6580 Torr. Perioperative arterial blood gases between control and shunted lambs were not remarkable or significantly different. Via a midsternotomy incision, the lambs were instrumented to measure vascular pressure and pulmonary blood flow. After 60 min of recovery, baseline hemodynamic variables and O2 saturation levels were obtained. Both total instrumentation time, 45 min, and total ventilation time,
2 h, were not significantly different between groups. The lambs were then euthanized with an intravenous injection of pentobarbital sodium (Euthanasia CII, Central City Medical, Union City, CA). The lung tissue was removed and prepared for Northern blot and protein analyses. All procedures and protocols were approved by the Committee on Animal Research of the University of California, San Francisco.
Measurements
Pulmonary and systemic arterial and right and left atrial pressures were measured by using Sorenson neonatal transducers (Abbott Critical Care Systems, Chicago, IL). Mean pressures were obtained by electrical integration. Heart rate was measured by a cardiotachometer triggered from the phasic systemic arterial pressure-pulse wave. Left pulmonary blood flow was measured on an ultrasonic flowmeter (Transonic Systems). All hemodynamic variables were recorded continuously on a multichannel electrostatic recorder (Gould, Cleveland, OH). Systemic arterial blood gases and pH were measured on a Radiometer ABL5 pH and blood-gas analyzer (Radiometer, Copenhagen, Denmark). Hemoglobin concentration and O2 saturation were measured by using a hemoximeter (model 270, Ciba-Corning). The pulmonary-to-systemic blood flow ratio was calculated by using the Fick principle (Table 1).
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Distal lung samples were excised, weighed, and snap-frozen in liquid N2. Samples were stored at 70°C until used for analysis.
Preparation of RNA, Northern Blotting, and Hybridization
Lung tissue was pulverized and then briefly homogenized in RNA-STAT (Tel-Test, Friendswood, TX). Total cellular RNA was extracted with phenol-chloroform, precipitated with isopropanol, and quantitated spectrophotometrically. RNA integrity was assessed by electrophoresis with ethidium bromide staining for rRNA. Total RNA (5 µg/lane) was separated electrophoretically on 1% agarose gels, transferred to nylon membranes by downward capillary action (Schleicher & Schuell, Keene, NH), and cross-linked with ultraviolet light (UV Stratalinker 2400, Stratagene). The blots were probed with cDNAs for ovine SP-A, SP-B, and SP-C (a kind gift from Dr. Phillip Ballard, University of Pennsylvania, Philadelphia, PA), labeled with [-32P]dCTP (NEN Research Products, Boston, MA) by random-primer second-strand synthesis (Random Primer Labeling Kit; GIBCO/BRL, Gaithersburg, MD). Filters were prehybridized for 30 min in QuikHyb Hybridization Solution (Stratagene, La Jolla, CA) at 42°C and then hybridized with probes at 1.251.5 x 106 disintegrations·min1·ml1 for 18 h. Hybridized filters were washed under high- and low-stringency conditions and subjected to autoradiography (Hyperfilm, Amersham, CEA, Uppsala, Sweden). Radiolabeled bands were quantified by volume integration of pixels measured by Phosphorimager analysis (ImageQuant Software; Molecular Dynamics, Sunnyvale, CA). Filters were probed for 18s rRNA as a control measure to ensure equal loading of samples.
Quantification of SP-A and SP-B by Dot Blot Analysis
Lung tissue was thawed at room temperature (RT) and homogenized in 50 mM NaHCO3, pH 9.0, buffer with protease inhibitors. The samples were sonicated on ice for 30 s and centrifuged at 14,000 rpm for 30 s. The supernatant was removed, and the protein content was measured by the bicinchoninic acid method (Pierce, Rockford, IL) (55). SP-A and SP-B were assayed by quantitative dot blotting. Dot blot of serial dilutions of control lambs was screened for antibodies for SP-A and SP-B proteins (Fig. 1). Comparison between control and shunted lambs was made among protein concentrations within the linear range. Standard curves were generated for both SP-A and SP-B. The supernatants (0.5 µg/well) were run in triplicates for SP-A and duplicates for SP-B on nitrocellulose (Bio-Dot Slot Format; Bio-Rad Laboratories, Richmond, CA). A gentle vacuum was applied to the dot blot apparatus until all of the solutions were pulled through. The nitrocellulose was incubated in 15% H2O2 for 15 min to quench any endogenous peroxidase activity, and nonspecific binding was blocked by incubation with a solution of 1% nonfat milk, 0.4% gelatin, 0.1% BSA, 0.9% NaCl, and 10 mM Tris-buffered saline (TBS) (pH 7.2) at RT for 1 h. The blots were then washed in TBS with 0.5% Tween 20 (TBS-T) for 5 min at RT. The blots were incubated in primary rabbit antibody (1:2,000 dilution) against ovine sheep SP-A and SP-B for 30 min. The blots were washed repeatedly in TBS-T over 1 h and then incubated in peroxidase-labeled donkey anti-rabbit secondary antibody (1:2,000 dilution; Amersham) for 20 min. Unbound secondary antibody was removed with TBS-T over 1 h. Bound secondary antibody was detected by exposure to luminol (ECL light detection system; Amersham) for 5 min and autoradiography. Relative light units were measured in a plate luminometer (Packard Instrument, Downers Grove, IL).
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Tissue samples were pulverized in liquid nitrogen by using a mortar and pestle, reconstituted in an extraction solvent containing double distilled water, chloroform, and methanol (0.8:2.0:1.0 vol/vol/vol), and shaken vigorously for 1 h at RT. By a modification of the Bligh-Dyer extraction technique (10), the chloroform phase containing all of the lipids was gently separated and evaporated by using a stream of nitrogen gas. The sample was reconstituted in a mixture of chloroform-methanol (2:1 vol/vol). A small aliquot was removed and dried under N2 gas. Spectrophotometric measurement of phosphorus level was made by using a modification of the Bartlett assay, which was used to determine total PL content per gram of tissue (6). An internal standard, L--diheptadecanoyl (diC17PC, Avanti Polar Lipid, Alabaster, AL), was added to the remaining solution, and an aliquot was spotted onto a silica plate for thin-layer chromatography. A Touchstone solvent (57) was used in the thin-layer chromatography to separate the PL classes. The spots were visualized by ultraviolet light by using a 0.25% 8-anilino-1-naphthalene-sulfonic acid spray and identified by their migration distance vs. the standard. The spots were then scraped and extracted by using the Bligh-Dyer method. Another internal standard, methyl nonadecanoate (C19FAME, Nu-CHEK Prep, Elysian, MN), was added to the chloroform phase containing the PL and dried under N2 gas. A 1% H2SO4 solution in methanol was added to the samples before they were heated at 70°C for 1 h. The fatty acids associated with the PL were esterified to fatty acid methyl esters and extracted with water and n-heptane. Three heptane fractions were pooled and dried under N2 gas. Each sample was reconstituted in hexane and injected into a gas chromatograph, whereby the fatty acid chains were separated by length and saturation. From the peak areas in the gas chromatograph, the minimum percentage of saturated phosphatidylcholine (PC) was determined, assuming that no diunsaturated fatty acids were present (1).
DNA Analysis
The DNA determination method described by Setaro and Morley (53) was used. DNA standards (50 µg calf thymus DNA/ml of 1 N NH4OH; Sigma no. D1501) and samples were dried at 60°C with N2 for 510 min. One hundred microliters of 3,5-diaminobenzoic acid (Sigma no. D1891, 0.3 g/ml double distilled water) were added to each tube, vortexed, incubated at 60°C for 5 min, vortexed, and then incubated again for 40 min. The samples were cooled to RT for 5 min, and 1.4 ml of 1 N HCl were added to each tube, vortexed, and read by a fluorimeter with excitation 410 nm, emission 520 nm, and slit width 0.5 mm (Fluorolog, ISA Instruments, Research Triangle Park, NC). Values were normalized to gram of lung weight.
Statistical Analysis
Comparisons between shunt and age-matched controls were made by using an unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Shunted lambs were similar in age as controls on the day of the experiment (5.4 ± 3.1 vs. 5.4 ± 2.8 days old). Shunted lambs had a mean pulmonary arterial pressure of 28.0 ± 8.5 mmHg, 61% of systemic values, compared with 15.5 ± 2.2 mmHg, 28% of systemic values, for control lambs (P < 0.05). By the Fick equation, shunted lambs had a pulmonary-to-systemic blood flow ratio of 2.7 ± 0.7. In addition, left pulmonary blood flow (115.7 ± 28.9 vs. 51.3 ± 22.8 ml·kg1·min1, P < 0.05) and left atrial pressure (10.2 ± 3.8 vs. 5.1 ± 1.8 mmHg, P < 0.05) were higher among shunted lambs. In contrast, the mean systemic arterial pressure (46.0 ± 3.7 vs. 54.6 ± 10.2 mmHg, P < 0.05) and left pulmonary vascular resistance (0.174 ± 0.14 vs. 0.250 ± 0.14 mmHg·ml1·min·kg, P < 0.05) were lower among shunted lambs. There was no statistical difference in weight, heart rate, and right atrial pressure between animal groups, although weight and right atrial pressure tended to be higher among shunted lambs (Table 1).
Effects of Increased Pulmonary Blood Flow on SP-A, SP-B, and SP-C mRNA Contents
SP-A, SP-B, and SP-C mRNA contents, as determined by Northern Blot analysis, were normalized to 18S rRNA. Shunted lambs had a decrease in SP-A mRNA content of 9.4% (2.21 ± 0.35 vs. 2.44 ± 0.50, P = 0.37, n = 8 for shunted and n = 8 for control), SP-B mRNA content of 10.8% (10.37 ± 2.29 vs. 11.62 ± 2.17, P = 0.30, n = 8 shunted and n = 8 control), and SP-C mRNA content of 13.1% (6.95 ± 1.10 vs. 7.99 ± 1.80, P = 0.33, n = 8 shunted and n = 8 control) of age-matched controls, which were not statistically significant (Fig. 2).
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On dot blot analysis, SP-A protein content, as standardized to grams of lung weight, was decreased by 7.4% compared with age-matched controls, but did not reach statistical significance (8.44 x 106 ± 1.14 x 106 vs. 9.11 x 106 ± 9.00 x 105, P = 0.33, n = 8 for shunted and n = 8 for control). There was an increase in SP-B protein content in shunted animals of 12.4% (6.49 x 106 ± 7.78 x 105 vs. 5.77 x 106 ± 6.48 x 105, P = 0.42, n = 8 for shunted and n = 8 for control) compared with age-matched controls, which was not statistically different between the two groups (Fig. 3).
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Total PL levels were similar between control and shunted lambs (15.90 ± 5.42 vs. 15.20 ± 4.04 mg PL/g wet wt, P = 0.39, n = 7 for control and shunted lambs). There was no significant difference in %PC in total PL (32.78 ± 10.03 vs. 30.21 ± 7.44%, P = 0.29, n = 7 for control and shunted) nor %saturated PC levels (46.53 ± 7.75 vs. 48.59 ± 4.29%, P = 0.30, n = 7 for control and shunted) (Table 2).
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To determine whether changes in SP mRNAs were due to changes in cell numbers, the mean DNA content was measured between shunted and control lambs. There was no statistical difference between the groups (Fig. 4).
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DISCUSSION |
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Previously, in our laboratory's model (52) of congenital heart disease in lambs at 1 wk of age, the increase in pulmonary blood flow was associated with an increased ET-1 activity, without a change in NO level or function (47). By 4 wk of age, there was an increase in endothelial NO synthase (NOS; eNOS) mRNA and protein levels and increased ET-1 mRNA and protein contents (8, 9). Corresponding with the changes in the vasoactive mediators, there was a decrease in SP-A mRNA as well as SP-A and SP-B protein contents (25). The increase in eNOS transcription was lost by 8 wk of age (8, 9). Presently, we have found a decrease in SP-A gene mRNA transcription of 9.4% and protein content of 7.4% within the first week of life between control and shunted lambs, which was not significant but which seemed to follow the trend seen at 4 wk of age. Both SP-B and SP-C mRNA transcription were also decreased by 10.8 and 13.1%, respectively, whereas SP-B protein content was increased by 12.4%; none of the changes was statistically significant. To determine whether the effect of increased pulmonary blood flow was on lipid composition, specifically PC, the main constituent of surfactant, total PL, %PC in PL, and %saturated PC were measured. There was no significant difference between control and shunted lambs in lipid composition. Our study reinforced the observation that increased pulmonary flow affected both endothelial and epithelial cell function in vivo and alluded to a possible relationship between NO and surfactant.
The relationship between NO and surfactant within the first week of life to 4 wk of age raises an interesting question of whether or not NO is involved in the regulation of surfactant synthesis, secretion, and/or metabolism. Multiple studies involving NO have produced seemingly conflicting results. Recently, Stuart et al. (56) found that ventilating 4-wk-old lambs with exogenous inhaled NO at 40 ppm increased SP-A and SP-B mRNA levels at both 12 and 24 h with a corresponding increase in SP-A and SP-B protein contents at 12 h. However, by 24 h, there was a decrease in SP-A and SP-B protein contents of 70 and 65%, respectively (56). In contrast, Ayad and Wong (3) showed that in vitro exposure to an NO donor, S-nitroso-N-acetyl penicillamine, decreased SP-A mRNA and protein content in a human lung tumor cell line representative of distal respiratory epithelium. Haddad et al. (28) demonstrated that, in freshly isolated rat alveolar type II cell, several NO donors decreased the rate of surfactant synthesis as measured by the incorporation of methyl-3H into PC and a reduction in cellular ATP levels. In this same study, however, ventilating the rats with inhaled NO at 80 ppm did not affect surfactant synthesis or ATP levels (28). In our study, shunted lambs had no statistical change in surfactant gene expression within the first week of life, although there was a reduction in SP-A mRNA and protein content. By 4 wk of age, an increase in NO level was associated with a decrease in SP-A mRNA level and SP-A and SP-B protein contents. Consequently, increased pulmonary blood flow may stimulate endothelial NO synthesis, which then may decrease the synthesis of surfactant.
Several studies have also suggested a protective role in inhibiting NOS on surfactant function in models of acute lung injury and/or acute respiratory distress syndrome (7, 35, 38, 50, 58). McDowell et al. (44) showed that mice, in an acute lung injury model caused by nickel, treated with NG-nitro-L-arginine methyl ester (l-NAME), a NOS inhibitor, had restored levels of surfactant SP-A, SP-B, and SP-C mRNAs, which were previously decreased. Cruz et al. (19), in a rat model of acute respiratory distress syndrome caused by N-nitroso-N-methylurethane, also showed that l-NAME normalized the alveolar-arterial oxygen tension, attenuated the decrease in PL-to-protein ratio, elevated minimal surface tension of crude surfactant pellet caused by N-nitroso-N-methylurethane, and decreased the infiltration of neutrophils into the alveolar space. In both studies, the protective effect of l-NAME seemed to be associated with its effect on eNOS, not inducible NOS. Interestingly, in a model of acute lung injury from sepsis and hyperoxia, there was no difference in total surfactant levels or large aggregates in lavages between wild and inducible NOS (/) knockout mice (4). The conflicting results between in vivo and in vitro studies may be explained by the presence of various antioxidants within epithelial lining fluid in vivo not present in vitro, which inactivates the toxic intermediates of NO, such as peroxynitrite (26, 27, 29, 43, 60), methemoglobin (30, 31, 33), and nitrogen dioxide (45), which affect SP synthesis, secretion, or function.
Whether or not alveolar epithelial lining fluid contains antioxidants in vivo, which prevents NO-induced surfactant dysfunction among in vitro cells, will need to be further elucidated. Interestingly, in the study by Stuart et al. (56), the SP-A and SP-B exposed to NO decreased by 24 h. One wonders whether nitration of tyrosine residues increased SP metabolism and degradation.
In conclusion, our study showed no changes in surfactant mRNA or protein content within the first week of life among lambs with congenital heart disease associated with increased pulmonary blood flow. This contrasted with a corresponding group of lambs at 4 wk of age that did show a decrease in surfactant SP-A mRNA transcription and SP-A and SP-B protein content associated with an increase in NOS not seen within the first week of life. These changes may be adaptive measures to the progressive structural changes seen in the pulmonary vasculature. The role of SPs in relation to vasoactive mediators in the development of pulmonary vascular resistance must be further elucidated. A limitation to the present study is the lack of investigation into the change in SP-D with increased pulmonary blood flow. SP-A and SP-D are lung collectins, which are important to innate immune response to microbial challenge as well as to inflammatory regulation within the lung. The changes in the lung collectins may play a significant role in the contribution of pulmonary complications to perioperative morbidity to repair (2, 18, 4042). However, the results seem to support what is now seen clinically in the surgical literature: improved outcomes with early surgical repair in neonates with congenital heart disease with significant left to right shunts, as opposed to palliation and delayed repair (13, 14, 39, 51). Early surgical intervention may bypass the progressive changes in surfactant, vasoactive substances such as NO and ET-1, and pulmonary vasculature, which increases the morbidity and mortality of late repair.
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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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