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Am J Physiol Lung Cell Mol Physiol 281: L1264-L1270, 2001;
1040-0605/01 $5.00
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Vol. 281, Issue 5, L1264-L1270, November 2001

Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow

Jorge A. Gutierrez1, Andrew J. Parry2, D. Michael McMullan2, Cheryl J. Chapin1, and Jeffrey R. Fineman1,3

Departments of 1 Pediatrics and 2 Surgery and 3 Cardiovascular Research Institute, University of California, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Infants with increased pulmonary blood flow secondary to congenital heart disease suffer from tachypnea, dyspnea, and recurrent pulmonary infections. We have recently established a model of pulmonary hypertension secondary to increased pulmonary blood flow in lambs after in utero placement of an aortopulmonary vascular graft. The purpose of the present study was to utilize our animal model to determine the effects on the expression of surfactant proteins A (SP-A), B (SP-B), and C (SP-C). At age 4 wk, SP-A mRNA content in lambs decreased to 61.4 ± 8% of age-matched control value (n = 5; P < 0.05). In addition, SP-A protein content was decreased to 50 ± 12% of control value (n = 6; P < 0.0001). Although we did not observe statistically significant changes in SP-B mRNA content, SP-B protein was decreased to 74 ± 25% of control value (n = 4; P < 0.02). There was no difference in SP-C mRNA. These data show that in a model of congenital heart disease with pulmonary hypertension secondary to increased pulmonary blood flow, there is a decrease in SP-A gene expression as well as a decrease in SP-A and SP-B protein contents.

congenital heart disease; gene expression; overcirculation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ALVEOLAR FLUID is composed of surfactant, which is a complex lipoprotein that is assembled and secreted into the alveolar spaces by alveolar epithelial type II cells. The composition of surfactant is ~90% lipids and ~10% proteins. The main lipid fraction is the saturated lecithin dipalmitoylphosphatidylcholine (DPPC). Four lipid-associated apolipoproteins have been isolated from lung surfactant and are designated surfactant proteins A, B, C, and D (SP-A, SP-B, SP-C, and SP-D, respectively). The primary function of lung surfactant is to lower surface tension at the air-water interface of the lung alveoli, thereby stabilizing lung volume at low transpulmonary pressures. Deficiencies of surfactant are known to result in decreased lung compliance and respiratory failure (1, 7). In addition, recent studies demonstrate that surfactant plays an important role in pulmonary host defense (21, 32, 35).

The lung is a dynamic organ subjected to varying mechanical forces throughout life. During fetal development, the lung is subjected to both tonic distension and fetal breathing movements. Alteration in these physical forces during development by either under- or overdistension results in profound abnormalities of fetal lung growth and surfactant maturation (20, 37). We have previously shown that mechanical forces are potent regulators of surfactant protein gene expression (15). In these studies, mechanical distension resulted in a decrease in mRNA content of SP-B and SP-C. These changes were found to be due to alterations at the transcriptional level. Recent data suggest that increased pulmonary blood flow and/or pulmonary vascular pressure may influence the components of the alveolar fluid (13). Although in vivo and in vitro data suggest that mechanical forces alter the expression of surfactant protein in the lung (15, 30), the effects of increased pulmonary blood flow and pulmonary hypertension on pulmonary alveolar epithelial function have not been investigated. The objective of the present study was to utilize our lamb model of congenital heart disease with increased pulmonary blood flow to determine the effects on the expression of SP-A, SP-B, and SP-C in vivo.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Surgical Preparations and Care

Ewes. Six mixed-breed, pregnant Western ewes carrying twins (137-141 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 in length; W. L. Gore, Milpitas, CA) was anastomosed between the ascending aorta and the main pulmonary artery of one twin as previously described (33). The ewe was returned to the cage after recovery from anesthesia and was given free access to food and water. Antibiotics (2 × 106 U of penicillin G potassium and 100 mg of gentamicin sulfate) were administered to the ewe during surgery and daily thereafter. Two twin controls underwent a sham thoracotomy without placement of the anastamosis. The remaining twins were exposed to hysterotomy but did not undergo sham thoracotomy. We did not detect differences between these control groups (Table 1).

                              
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  		Table 1.   Effects of hysterotomy and sham thoracotomy

Lambs. After spontaneous delivery, antibiotics (1 × 106 U of penicillin G potassium and 25 mg of gentamicin sulfate im) were administered for 2 days. Furosemide (1 mg/kg im) was administered daily. Elemental iron (50 mg im) was given weekly. While under local anesthesia with 1% lidocaine hydrochloride, 1-mo-old lambs had polyvinyl catheters placed in an artery and vein of one hind leg. These catheters were advanced to the descending aorta and the inferior vena cava, respectively. The lambs were then anesthetized with intravenous infusions of ketamine hydrochloride (~1 mg · kg-1 · min-1) and diazepam (0.002 mg · kg-1 · h-1), intubated with a 5.5-mm-OD endotracheal tube, and mechanically ventilated with a pediatric, time-cycled, pressure-limited ventilator (Healthdyne, Marietta, GA). Heart rate and systemic blood pressure were monitored continuously to ensure adequate anesthesia. Ventilation with a peak inflating pressure of 25 cmH2O and an end-expiratory pressure of 5 cmH2O and with 21% O2 was adjusted to maintain an arterial PCO2 (PaCO2) between 35 and 45 Torr. A midsternotomy incision was performed, and the pericardium was incised. Three single-lumen polyurethane catheters were inserted into the left and right atria and the main pulmonary artery. An ultrasonic flow probe (Transonics Systems, Ithaca, NY) was placed around the left pulmonary artery to measure left pulmonary blood flow. After 60 min of recovery, baseline hemodynamic variables and O2 saturation levels were obtained. 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 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 using a hemoximeter (model 270, Ciba-Corning). The pulmonary-to-systemic blood flow ratio was calculated using the Fick principle.

Lung Tissue Preparation

Distal lung samples were excised, weighed, and snap-frozen in liquid N2. Samples were stored at -70°C until used for analysis.

Protein Determination

Protein content was measured via the bicinchoninic acid method (Pierce, Rockford, IL).

Quantification of SP-A by dot blotting. Western blot analysis was performed on protein samples from control and shunted lambs to ascertain which protein species were detectable from homogenates of whole sheep lung. Samples (10 and 5 µg) and prestained molecular mass standards (GIBCO BRL, Gaithersburg, MD) were electrophoresed under reducing conditions through a 4% acrylamide stacking gel and subsequent 15% polyacrylamide gel (Fig. 1). Proteins were electrophoretically transferred to nitrocellulose paper. Gel protein transfer was confirmed by absent Coomassie blue staining. Western blots were blocked for 2 h in a solution of 1% nonfat dried milk, 0.4% gelatin, and 0.1% BSA in 150 mM NaCl-10 mM Tris · HCl (pH 7.2). Blots were incubated for 1 h in 20 mM Tris-buffered saline (TBS, pH 7.4) containing anti-SP-A no. 1767 (a 1:5,000 dilution), which is a polyclonal antibody against ovine SP-A (a kind gift from Dr. Sam Hawgood, University of California, San Francisco). After 20 washes with TBS, the blots were incubated for 30 min in a 1:3,000 dilution of horseradish peroxidase-labeled, affinity-purified donkey anti-rabbit IgG (Amersham, Uppsala, Sweden). Blots were washed again and then incubated in ECL Plus reagent (Amersham) for 5 min.


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  		Fig. 1.   Western blot of sheep whole lung homogenate [10 (left control and left shunt lanes) and 5 (right control and right shunt lanes) µg of total protein] and purified sheep surfactant protein A (SP-A; 0.05 µg) probed with polyclonal anti-sheep SP-A antibody no. 1767. Bands of the appropriate molecular mass were detected in whole lung homogenates; antibody also detected degraded products.

SP-A was assayed by quantitative dot blotting (Fig. 2). Duplicate dots of serial dilutions of samples and purified sheep SP-A (a kind gift from Dr. Joe Kitterman, University of California, San Francisco) were assayed on the same piece of nitrocellulose. Results are expressed in micrograms of SP-A and were normalized to dry lung weight. Protein samples from control and shunted animals were diluted 1:100 with 50 mM NaHCO3 (pH 9.0) and dot blotted onto nitrocellulose. Endogenous peroxidase activity was quenched by treatment with 15% hydrogen peroxide for 5 min, and nonspecific binding was blocked by a 1-h incubation in a solution of 1% nonfat milk, 0.4% gelatin, 0.1% BSA, 0.9% NaCl, and 10 mM TBS (pH 7.2). Primary antibody to ovine SP-A (a kind gift from Dr. Sam Hawgood, University of California, San Francisco) in blocking buffer (a 1:5,000 dilution) was then incubated with the blot for 20 min. The blot was washed 20 times with 20 mM TBS (pH 7.4) containing 0.05% Tween (TBS-T) and then incubated with a solution containing peroxidase-labeled donkey anti-rabbit secondary antibody (Amersham) in TBS-T (a 1:5,000 dilution). After a 20-min incubation, unbound secondary antibody was removed by 10 washes in TBS-T. Bound secondary antibody was detected by exposure to luminol (ECL light detection system, Amersham) for 1 min and autoradiography. Relative light units were measured in a plate luminometer (Packard Instrument, Downers Grove, IL).


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  		Fig. 2.   Dot blot of serial dilutions of purified SP-A and the resulting linear portion of the standard curve. Amounts of SP-A in each sample were derived by comparing relative light units (RLU) in the linear portion of serial dilutions of the sample to the purified SP-A standard curve. Blots were incubated with primary (1°) and secondary (2°) antibodies as well as secondary antibody alone to ensure specificity of the secondary antibody.

Determination of SP-B protein. Western blot analysis was performed on protein samples from control and shunted lambs under nonreducing conditions as described (see Quantification of SP-A by dot blotting). Western blots were incubated with anti-SP-B no. 1768 (a 1:5,000 dilution), which is a polyclonal antibody against ovine SP-B (a kind gift from Dr. Sam Hawgood, University of California, San Francisco).

Quantitative dot blot analysis was performed on serial dilutions of protein homogenates from control and shunted lambs. Dots were quantitated by phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA). Due to the lack of purified SP-B, data are presented as percent of control.

Preparation of RNA, Northern Blotting, and Hybridization

Lung tissue was pulverized and 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 and ethidium bromide staining for rRNA. Total RNA (10 µg/sample) was separated electrophoretically on 1% agarose gels before it was transferred to nylon membranes under positive pressure (Posiblotter, Stratagene, La Jolla, CA) and cross-linked with ultraviolet light (UV Stratalinker 2400, Stratagene). Filters were probed with cDNAs for ovine SP-A, SP-B, SP-C (a kind gift from Dr. Phillip Ballard, University of Pennsylvania, Philadelphia, PA), and 18S rRNA, and were labeled with [alpha -32P]dCTP (NEN Research Products, Boston, MA) by random primer second-strand synthesis (random primer labeling kit, GIBCO BRL). Filters were prehybridized for 10 min in QuikHyb hybridization solution (Stratagene) at 68°C and then hybridized in 10 ml of QuikHyb solution containing 1.25 × 106 dpm/ml for 18 h. Hybridized filters were washed under high-stringency conditions and subjected to autoradiography (Hyperfilm, Amersham) before radiolabeled bands were quantified by volume integration of pixels measured by phosphorimager analysis (ImageQuant software, Molecular Dynamics). Using 18S rRNA as a control ensured equal loading.

Statistical Analysis

Comparisons between shunt and age-matched controls were made using ANOVA or an unpaired t-test. P < 0.05 was considered significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of Hysterotomy and Sham Thoracotomy

There were no differences between animals that had undergone sham surgery (thoracotomy without aortopulmonary graft placement) and nonsham controls (hysterotomy only) in weight, pulmonary arterial pressure, systemic blood pressure, heart rate, pH, PCO2, and SP-A and SP-B protein contents (Table 1).

Effects of Increased Pulmonary Blood Flow on Hemodynamics, Lung Water, and Total Protein

Shunted lambs had a mean pulmonary arterial pressure of 48.3 ± 11.7 mmHg, which represents 78% of systemic values. In comparison, control animals had a mean pulmonary arterial pressure of 15.3 ± 3.7 mmHg, which represents 23% of systemic values (n = 6; P < 0.05). Shunted animals had a pulmonary-to-systemic blood flow ratio of 2.25 ± 1.3 as determined by the Fick equation. In addition, left pulmonary blood flow (1.54 ± 0.66 vs. 0.50 ± 0.16 l/min; P < 0.05), left atrial pressure (12.6 ± 4.7 vs. 7.1 ± 2.0 mmHg; P < 0.05), and right atrial pressure (6.8 ± 3.0 vs. 3.3 ± 2.3 mmHg; P < 0.05) were increased in shunted lambs. Mean systemic arterial pressure (62.0 ± 7.8 vs. 72.0 ± 9.2 mmHg; P < 0.05) was decreased in shunted lambs, and heart rate (142.3 ± 21.6 vs. 141.6 ± 25.6 beats/min) was unchanged. There was no difference in birth weight between shunted and control lambs, although shunted lambs weighed less at 4 wk of age than controls (8.1 ± 2.1 vs. 14.2 ± 3.1 kg; P < 0.05).

Shunted lambs had increased lung water as exhibited by dry lung weight as a percent of wet lung weight: 4.7 ± 2% compared with 9.3 ± 2% for controls (n = 6; P < 0.05). Total protein, measured as milligrams of protein per gram of dry lung weight, was decreased in the shunted animals; however, these differences (controls, 149 ± 48 mg/g dry lung; shunted animals, 125 ± 27 mg/g dry lung) were not statistically significant.

Effects of Increased Pulmonary Blood Flow on SP-A

SP-A protein content was standardized to grams of dry lung weight. Shunted animals had a decrease in SP-A protein content to 50 ± 12% of age-matched controls (n = 6; P < 0.0001; Fig. 3). SP-A mRNA content as determined by Northern blot analysis was normalized to 18S rRNA. Shunted lambs had a decrease in SP-A mRNA content to 61 ± 8% of control value (n = 5; P < 0.05; Fig. 4).


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  		Fig. 3.   SP-A protein levels in control (C) and shunted (S) lambs. Quantitative dot blots were performed on whole lung homogenates as described in text. Amount of SP-A was determined and standardized to grams of dry lung weight. Values are means ± SD of 5 experiments. *P < 0.0001.



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  		Fig. 4.   Effects of increased pulmonary blood flow on SP-A, SP-B, and SP-C mRNA contents. Bottom: Northern blots were performed from RNA obtained from control (open bars) and shunted (solid bars) lambs as described in text. Autoradiograms were obtained before radioactivity was quantified via phosphorimager analysis, and 18S protein rRNA was used as a control for equal loading of RNA into each lane. Top: left 2 lanes/blot, duplicate control samples; right 2 lanes/blot, duplicate shunt samples. Results are means ± SD in percent change from control values from 5 experiments. *P < 0.05.

Effects of Increased Pulmonary Blood Flow on SP-B

Homogenates of lung from control and shunted lambs were analyzed by Western blot analysis. Anti-SP-B antibody reacted with unreduced proteins with apparent molecular masses of 40-42, 28, 26, and 18 kDa. The detection of the SP-B precursor, processing intermediate, and homodimer at 40-42, 28, and 26 kDa, respectively, has previously been demonstrated (6) for the mouse (Fig. 5). Using dot blot analysis, SP-B protein levels were decreased in shunted lambs to 74 ± 25% of control value (n = 4; P < 0.02; Fig. 5).


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  		Fig. 5.   Analysis of SP-B protein from control (open bar) and shunted (solid bar) lambs. Protein samples [15 (right shunt and right control lanes) and 30 (left shunt and left control lanes) µg] from whole lung homogenates were loaded onto adjacent lanes of 15% SDS-polyacrylamide gels, separated by electrophoresis, and then transferred to nitrocellulose membranes. Membranes were subjected to Western blot analysis using anti-SP-B antibody. Bands were revealed using ECL Plus reagents. Precursor protein was detected at ~43 kDa, a processing intermediate was revealed at ~28 and 26 kDa, and the fully processed form was indicated at ~18 kDa. Quantitative dot blots were performed on whole lung homogenates as described in text. Results are means ± SD in percent of control values from 4 experiments. *P < 0.02.

SP-B mRNA content was determined by Northern blot analysis and normalized to 18S rRNA. Both previously reported splice mRNA species were detected (31). There was no difference in SP-B mRNA content in shunted lambs compared with control lambs (95 ± 20%, n = 5; P = 0.45; see Fig. 4).

Effects of Increased Pulmonary Blood Flow on SP-C mRNA.

SP-C mRNA content was determined by Northern blot analysis. There was no statistical difference in SP-C mRNA content in shunted lambs compared with control lambs (115 ± 10%, n = 5; P = 0.60; see Fig. 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Increased pulmonary blood flow secondary to congenital heart disease produces increases in vascular shear stress, which results in profound effects on vascular endothelial cell function. For example, shear stress stimulates the synthesis of mRNA for platelet-derived growth factor-A and -B (16, 30), tissue plasminogen activator (9), and intercellular adhesion molecule-1 (26) in human vascular endothelial cells. Increases in shear stress also stimulate endothelial cells to produce several mediators of vascular tone including nitric oxide (19). However, the effects of increased pulmonary blood flow on alveolar epithelial cell function had not been previously studied. The present study is the first demonstration that increased pulmonary blood flow and/or pressure can affect surfactant protein expression. In a lamb model of congenital heart disease with increased pulmonary blood flow and pressure, we found that SP-A mRNA content was decreased to 61% of control value, and SP-A and SP-B protein contents were decreased to 50 and 74%, respectively, of control value.

The mechanisms by which increased pulmonary blood flow and/or pressure decrease surfactant protein expression are speculative at this time. Pulmonary alveolar epithelial cells are known to be responsive to mechanical forces in vivo (30) and in vitro (15). Surfactant proteins have been shown to be regulated at the transcriptional level by mechanical forces (14, 15). It is therefore possible that the changes in SP-A mRNA and protein content are in direct response to changes in shear stress in the pulmonary vasculature. SP-A expression may also be altered by mediators of vascular tone, which are known to be regulated by shear stress. Nitric oxide is an important effector molecule that plays a central role in a number of physiological and pathological processes (18). Nitric oxide, which is increased in response to shear stress (19), easily diffuses into the alveolar space and has also been shown to modulate the expression of a number of genes. Recent data demonstrate that nitric oxide inhibits both surfactant synthesis in primary cultures of pulmonary alveolar type II cells (16) and expression of SP-A in a human pulmonary epithelial cell line (2). The shunted lambs in this study are known to have had increased expression of endothelial nitric oxide synthase mRNA and protein, cGMP, and nitrates (4) compared with control animals. It is therefore possible that increased pulmonary blood flow stimulates endothelial cell nitric oxide production, which then decreases expression of SP-A by alveolar type II cells.

There are also non-perfusion-related stimuli that can alter surfactant protein expression or secretion. For example, lung expansion has been shown to decrease surfactant protein mRNA levels in fetal sheep (24), and hyperventilation has been shown to increase the amount of phospholipid in bronchoalveolar lavage fluid (29). In addition, Massaro and Massaro (25) showed morphological evidence that deep inflation induced surfactant release by showing a decrease in the number of lamellar bodies in type II cells after deep inflation.

We were able to demonstrate a difference in SP-B protein content between control and shunted lambs, but we did not detect a statistically significant difference in mRNA content. There are a number of possible reasons for this observation. First, it is possible that the small difference in SP-B mRNA content, although not statistically significant, may have resulted in the change noted at the protein level. Second, we may have missed larger differences by only assessing mRNA content at the end of the study period. Finally, the observed differences may have been due to leakage of SP-B protein into the circulation, which occurs in the setting of increased alveolar permeability secondary to acute respiratory distress syndrome or acute cardiogenic pulmonary edema (10) and may not reflect an alteration in gene expression.

Interestingly, in contrast to SP-B and SP-C mRNA content, we did observe differences in SP-A mRNA content between shunted and control lambs. This apparent regulation of SP-A gene expression, which is different from SP-B and SP-C expression, has been observed previously. For example, in the developing lung, agents that increase intracellular cAMP concentration cause an increase in SP-A mRNA but have only a modest effect on the mRNA contents of SP-B and SP-C (23, 27). In human fetal lung tissue in vitro, glucocorticoids exert a marked stimulatory effect on the levels of SP-B and SP-C mRNAs (28, 36), whereas the effects on levels of SP-A mRNA are both stimulatory (at low concentrations) and inhibitory (at high concentrations) (5).

Children with congenital heart disease and increased pulmonary blood flow are known to have decreased lung compliance, increased expiratory airway resistance, and recurrent pulmonary infections (8, 11, 12, 38). Pulmonary surfactant proteins are important components of lung compliance and pulmonary immunity. However, we recognize that the magnitude of change in SP-A gene expression and SP-A and SP-B protein contents demonstrated in this study is relatively small. It is therefore difficult to make strong conclusions about the biological significance of these findings. Although SP-A-deficient mice have normal lung anatomy, function, and in vivo surfactant function, recent data demonstrate that these mice are susceptible to group B streptococcal infection (33) and Pseudomonas aeruginosa infection (22). In vitro, SP-A stimulates chemotaxis of alveolar macrophages (38), enhances complement-mediated phagocytosis (35), and binds and neutralizes influenza A viruses (3). We have demonstrated quantifiable changes in both SP-A mRNA and SP-A and SP-B protein contents that could be of biological relevance to the host defense capacities and surface-active properties of pulmonary surfactant.

Some limitations of this study are noteworthy. First, with this current model system, it is not possible to differentiate the potential effects of increased flow and increased pressure. Second, we were unable to control respiratory rate in the lambs while they were breathing spontaneously, and it is possible that increased respiratory rate secondary to increased lung water may have played a role in altering surfactant protein expression in this model. Although hyperventilation is known to cause increased surfactant secretion, its effect on surfactant protein gene expression remains unclear. In one previously published report (40), hyperventilation induced by exposing rats to a gas mixture of 5% CO2-13% O2-82% N2 did not alter the mRNA content of SP-A, SP-B, or SP-C in type II cells, but expression of SP-A and SP-B mRNA was increased in lung tissue. In view of these limitations, it is difficult to attribute causality to the observed changes based on these studies. We are currently evaluating a model of increased flow without increased pulmonary arterial pressure or hyperventilation to delineate the potential mechanism involved in the changes observed in this study.

In conclusion, we present evidence demonstrating that increased pulmonary blood flow and/or pressure decreases SP-A mRNA content and SP-A and SP-B protein levels in a lamb model of congenital heart disease. The mechanisms by which increased blood flow and/or pressure decrease surfactant protein expression remain to be elucidated. Further investigation into the mechanisms of these alterations may lead to a better understanding of the pulmonary aberrations noted in children with congenital heart disease and increased pulmonary blood flow.


    ACKNOWLEDGEMENTS

We thank Michael J. Johengen, Janine M. Bekker, and Wen Zhou for technical assistance.


    FOOTNOTES

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-61284 and HL-04372-01 and Robert Wood Johnson Foundation Grant 030805.

Address for reprint requests and other correspondence: J. A. Gutierrez, Dept. of Pediatrics, Univ. of California, San Francisco, 3333 California St., Box 1245, San Francisco, CA 94118-1245 (E-mail: jgut{at}itsa.ucsf.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 22 January 2001; accepted in final form 2 July 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Avery, ME, and Mead J. Surface properties in relation to atelectasis and hyaline membrane disease. Am J Dis Child 97: 517-523, 1959.

2.   Ayed, O, and Wong HR. Nitric oxide decreases surfactant protein A gene expression in H441 cells. Crit Care Med 26: 1277-1282, 1998[ISI][Medline].

3.   Benne, CA, Kraaijeveld CA, van Strijp JAG, Brouwer E, Harmsen M, Verhoef J, van Golde LMG, and van Iwaarden JF. Interactions of surfactant protein A with influenza viruses: binding and neutralization. J Infect Dis 171: 335-341, 1995[ISI][Medline].

4.   Black, SM, Fineman JR, Steinhorn RH, Bristow J, and Soifer SJ. Increased endothelial nitric oxide synthase in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Heart Circ Physiol 275: H1643-H1651, 1998[Abstract/Free Full Text].

5.   Boggaram, V, Smith ME, and Mendelson CR. Regulation of expression of the gene encoding the major surfactant protein (SP-A) in human fetal lung in vitro. Disparate effects of glucocorticoids on transcription and on mRNA stability. J Biol Chem 264: 11421-11427, 1989[Abstract/Free Full Text].

6.   Chi, X, Garnier G, Hawgood S, and Colten HR. Identification of a novel alternately spliced mRNA of murine pulmonary surfactant protein B. Am J Respir Cell Mol Biol 19: 107-113, 1998[Abstract/Free Full Text].

7.   Clark, JC, Wert SE, Bachurski CJ, Stahlman MT, Stripp BR, Weaver TE, and Whitsett JA. Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice. Proc Natl Acad Sci USA 92: 7794-7798, 1995[Abstract/Free Full Text].

8.   Davies, CJ, Cooper SG, Fletcher ME, Hatch DJ, Helms PJ, Gordon I, and Stocks J. Total respiratory compliance in infants and young children with congenital heart disease. Pediatr Pulmonol 8: 155-161, 1990[ISI][Medline].

9.   Diamond, SL, Sharefkin JB, Dieffenbach C, Frasier-Scott KL, McIntire V, and Eskin SG. Tissue plasminogen activator messenger RNA levels increase in cultured human endothelial cells exposed to laminar shear stress. J Cell Physiol 143: 364-371, 1990[ISI][Medline].

10.   Doyle, IR, Bersten AD, and Nicholas TE. Surfactant proteins-A and -B are elevated in plasma of patients with acute respiratory failure. Am J Respir Crit Care Med 156: 1217-1229, 1997[Abstract/Free Full Text].

11.   Freezer, NJ, Lanteri CJ, and Sly PD. Effect of pulmonary blood flow on measurements of respiratory mechanics using the interrupter technique. J Appl Physiol 74: 1083-1088, 1993[Abstract/Free Full Text].

12.   Griffin, AJ, Ferrara JD, Lax JO, and Cassels DE. Pulmonary compliance and index of cardiovascular status in infancy. Am J Dis Child 123: 89-95, 1972[Medline].

13.   Grigg, J, Kleinert S, Woods RL, Thomas CJ, Vervaart P, Wilkinson JL, and Robertson CF. Alveolar epithelial lining fluid cellularity, protein, and endothelin-1 in children with congenital heart disease. Eur Respir J 9: 1381-1386, 1996[Abstract].

14.   Gutierrez, JA, Ertsey R, Scavo LM, Collins E, and Dobbs LG. Mechanical distention modulates alveolar epithelial cell phenotypic expression by transcriptional regulation. Am J Respir Cell Mol Biol 21: 223-229, 1999[Abstract/Free Full Text].

15.   Gutierrez, JA, Gonzalez RF, and Dobbs LG. Mechanical distension modulates pulmonary alveolar epithelial expression in vitro. Am J Physiol Lung Cell Mol Physiol 274: L196-L202, 1998[Abstract/Free Full Text].

16.   Haddad, IY, Zhu S, Crow J, Barefield E, Gadilhe T, and Matalon S. Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide. Am J Physiol Lung Cell Mol Physiol 270: L898-L906, 1996[Abstract/Free Full Text].

17.   Hseih, HJ, Li NQ, and Frangos JA. Shear stress increases endothelial platelet-derived growth factor mRNA levels. Am J Physiol Heart Circ Physiol 260: H642-H646, 1991[Abstract/Free Full Text].

18.   Ignarro, LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 65: 1-21, 1989[Free Full Text].

19.   Korenaga, R, Ando J, Tsuboi H, Yang W, Sakuma I, Toyo-oka T, and Kamiya A. Laminar flow stimulates ATP- and shear stress-dependent nitric oxide production in cultured bovine endothelial cells. Biochem Biophys Res Commun 198: 213-219, 1994[ISI][Medline].

20.   Laberge, JM, Sigalet DI, and Gutterman FM. Congenital diaphragmatic hernia. In: Hernia, edited by Nyhus LM, and Condon RE.. Philadelphia, PA: Lippincott, 1995, p. 555-566.

21.   Levine, AM, Bruno MD, Huelsman KM, Ross GF, Whitsett JA, and Korfhagen TR. Surfactant protein A-deficient mice are susceptible to group B streptococcal infection. J Immunol 158: 4336-4340, 1997[Abstract].

22.   Levine, AM, Kurak KE, Bruno MD, Stark JM, Whitsett JA, and Korfhagen TR. Surfactant protein A-deficient mice are susceptible to Pseudomonas aeruginosa infection. Am J Respir Cell Mol Biol 19: 700-708, 1998[Abstract/Free Full Text].

23.   Liley, HG, White RT, Warr RG, Benson BJ, Hawgood S, and Ballard PL. Regulation of messenger RNAs for the hydrophobic surfactant proteins in human lung. J Clin Invest 83: 1191-1197, 1989.

24.   Lines, A, Nardo L, Phillips ID, Possmayer F, and Hooper SB. Alterations in lung expansion affect surfactant protein A, B, and C mRNA levels in fetal sheep. Am J Physiol Lung Cell Mol Physiol 276: L239-L245, 1999[Abstract/Free Full Text].

25.   Massaro, GD, and Massaro D. Morphologic evidence that large inflations of the lung stimulate secretion of surfactant. Am Rev Respir Dis 127: 235-236, 1983[ISI][Medline].

26.   Nagel, T, Resnick N, Atkinson WJ, Dewey CF, and Gimbrone MA. Shear stress selectively upregulates intracellular adhesion molecule-1 expression in cultured human vascular endothelial cells. J Clin Invest 94: 885-891, 1994.

27.   Odom, MJ, Snyder JM, and Mendelson CR. Adenosine 3'5'-monophosphate analogs and beta-adrenergic agonists induce the synthesis of the major surfactant apoprotein in human fetal lung in vitro. Endocrinology 121: 1155-1163, 1987[Abstract].

28.   O'Reilly, MA, Clark JC, and Whitsett JA. Glucocorticoid enhances pulmonary surfactant B gene transcription. Am J Physiol Lung Cell Mol Physiol 260: L37-L43, 1991[Abstract/Free Full Text].

29.   Oyarzun, MJ, and Clements JA. Control of lung surfactant by ventilation, adrenergic mediators, and prostaglandin in the rabbit. Am Rev Respir Dis 117: 879-891, 1978[ISI][Medline].

30.   Piedboeuf, B, Laberge JM, Ghitulescu G, Gamache M, Petrov P, Belanger S, Chen M, Hashim E, and Possmayer F. Deleterious effect of tracheal obstruction on type II pneumocytes in fetal sheep. Pediatr Res 41: 473-479, 1997[ISI][Medline].

31.   Pietschmann, SM, and Pison U. cDNA cloning of ovine pulmonary SP-A, SP-B, and SP-C: isolation of two different sequences for SP-B. Am J Physiol Lung Cell Mol Physiol 278: L765-L778, 2000[Abstract/Free Full Text].

32.   Pison, U, Max M, Weibbach S, and Pietschmann S. Host defense capacities of pulmonary surfactant: evidence for nonsurfactant functions of the surfacant system. Eur J Clin Invest 24: 586-599, 1994[ISI][Medline].

33.   Reddy, VM, Meyrick B, Wong J, Khoor A, Liddicoat JR, Hanley FL, and Fineman JR. In utero placement of aortopulmonary shunts: a model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs. Circulation 92: 606-613, 1995[Abstract/Free Full Text].

34.   Resnick, N, Collins T, Atkinson W, Bonthron DT, Dewey CFJ, and Gimbrone MA. Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element. Proc Natl Acad Sci USA 90: 4591-4595, 1993[Abstract/Free Full Text].

35.   Tenner, AJ, Robinson SL, Borchelt J, and Wright JR. Human pulmonary surfactant protein (SP-A), a protein structurally homologous to C1q, can enhance FcR- and CR1- mediated phagocytosis. J Biol Chem 264: 13923-13928, 1989[Abstract/Free Full Text].

36.   Venkatesh, VC, Ianuzzi DM, Ertsy R, and Ballard PL. Differential glucocorticoid regulation of the pulmonary hydrophobic surfactant proteins SP-B and SP-C. Am J Respir Cell Mol Biol 8: 222-228, 1993.

37.   Wigglesworth, JS, Desai R, and Hislop AA. Fetal lung growth in congenital laryngeal atresia. Pediatr Pathol 7: 515-525, 1987[Medline].

38.   Wright, JR, and Youmans DC. Pulmonary surfactant protein A stimulates chemotaxis of alveolar macrophage. Am J Physiol Lung Cell Mol Physiol 264: L338-L344, 1993[Abstract/Free Full Text].

39.   Yau, K-I, Fang L-J, and Wu M-H. Lung mechanics in infants with left-to-right shunt congenital heart disease. Pediatr Pulmonol 21: 42-47, 1996[ISI][Medline].

40.   Yogalingam, G, Doyle IR, and Power JHT Expression and distribution of surfactant proteins and lysozyme after prolonged hyperpnea. Am J Physiol Lung Cell Mol Physiol 270: L320-L330, 1996[Abstract/Free Full Text].

Am J Physiol Lung Cell Mol Physiol 281(5):L1264-L1270
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