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* Division of Pulmonary Biology, Children's Hospital Medical
Center, Cincinnati, OH 45229-3039; and Edited by Michael J. Welsh, University of Iowa, College of
Medicine, Iowa City, IA, and approved March 19, 2001 (received for review October 20, 2000)
The surfactant protein C (SP-C) gene encodes an extremely
hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the
lung. To discern the role of SP-C in lung function, SP-C-deficient ( Pulmonary surfactant is a
complex mixture of lipids and proteins that reduces surface tension at
the air-liquid interface and prevents alveolar collapse during
respiration. Deficiencies of pulmonary surfactant because of premature
birth or surfactant inactivation from lung injury can result in a
lethal respiratory distress syndrome (RDS). Surfactant replacement by
instillation of bovine surfactant extracts has proven effective at
treating neonatal RDS (1, 2). Pulmonary surfactant is synthesized and
secreted into the alveolar lumen by specialized alveolar type II cells.
Four surfactant proteins (SP) with unique properties have been
identified in pulmonary surfactant and are termed SP-A, SP-B, SP-C, and
SP-D. SP-A and SP-D are relatively hydrophilic proteins and contribute
to innate defense of the lung (SP-A, SP-D) (3) and surfactant
homeostasis (SP-D) (4). SP-B and SP-C are hydrophobic proteins that
enhance surface-active properties of surfactant phospholipid films (5).
Of the surfactant proteins, SP-C is the most hydrophobic. The alveolar
form of SP-C is a 34- to 35-aa peptide that is proteolytically processed from a 21-kDa precursor protein (5). The sequence of the SP-C
peptide is highly conserved among mammalian species. The extremely
hydrophobic properties of SP-C are a result of a valine-rich region
within an extended 23-aa hydrophobic domain from residues 13-35.
Nineteen of these 23 aa are valine, leucine, or isoleucine. This
hydrophobic domain forms an SP-C expression is initiated early in the embryonic period of lung
formation, wherein SP-C transcripts are detected uniformly in
epithelial cells lining the primitive airways. As the branching tubules
elongate, SP-C expression is decreased in cells of the proximal
conducting portion of the lung and maintained in epithelial cells in
the periphery of the developing respiratory tubules (7). Ultimately,
the respiratory tubules form alveoli and SP-C mRNA is detected only in
alveolar type II cells of the mature lung (7).
Surfactant preparations containing phospholipids and SP-C are highly
surface active in vitro (8, 9). Surfactant replacement with
phospholipid preparations enriched in SP-C are highly effective in
treatment of respiratory distress (1). Bovine surfactant preparations
presently in clinical use contain phospholipids and are enriched in
SP-C (8). A synthetic surfactant composed of dipalmitoyl-phosphatidylcholine, phosphatidylglycerol, and recombinant human SP-C restored lung compliance in premature sheep, rabbit, and
adult animal models of acute lung injury (10-14). SP-C-based surfactant presently is being evaluated in clinical studies for the
treatment of adult respiratory distress syndrome. Although in
vitro and in vivo studies indicate that SP-C
contributes to surfactant function, whether SP-C is required for or
contributes to surfactant function in vivo has not been
determined. To discern the role of SP-C in the lung, we have
inactivated the SP-C gene in embryonic stem (ES) cells to produce mice
lacking SP-C.
Vector Construction.
A 129/J mouse genomic library was screened to identify genomic clones
of the SP-C gene homologous to the 129 derived ES cells. A 2.1-kb
BamHI fragment containing exons 2-6 of the SP-C gene was
used for modification of the gene. Sequence encoding the hydrophobic polyvaline domain of the SP-C peptide was interrupted by insertional mutagenesis with a 1.6-kb pGKneo gene cassette. This insertion provided
positive selection for targeted cells by growth in the neomycin
analogue G418. The 2.1-kb SP-C plasmid was digested with ApaLI, which cuts at a unique ApaLI site located
in the SP-C polyvaline domain. The ApaLI linker pGKneoBPA
cassette was ligated into the SP-C ApaLI site. A 1.3-kb
PstI-to-BamHI fragment spanning exon 1 and the 5'
flanking DNA was ligated to the 5' BamHI site of the 2.1-kb
Bam-pGKneoBPA fragment. The targeting construct was modified further by
cloning the herpes simplex virus thymidine kinase gene into the 5'
SphI site to provide gancyclovir selection against
nonhomologous integration of the construct.
Generation of SP-C Null Mutant Mice.
The D3R strain of ES cells (kind gift of Thomas Doetschman and John
Duffy, University of Cincinnati) was electroporated with the purified
SP-C-targeting construct DNA and selected as described (15). ES cell
DNA was digested with Bsu36I and analyzed with a probe
outside of the targeting construct sequence. The probe was a 457-bp
SphI-PstI fragment adjacent to the 5' limit of
the targeting construct. Positive clones were confirmed by genomic Southern blot of multiple restriction enzyme digests.
Medical Sciences
Altered stability of pulmonary surfactant in SP-C-deficient mice
,
, Division of
Clinical Science, Telethon Institute for Child Health Research, Perth
6872, Australia
Abstract
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
/) mice were produced. The SP-C (/) mice were viable at
birth and grew normally to adulthood without apparent pulmonary
abnormalities. SP-C mRNA was not detected in the lungs of SP-C
(/) mice, nor was mature SP-C protein detected by Western blot of
alveolar lavage from SP-C (/) mice. The levels of the other
surfactant proteins (A, B, D) in alveolar lavage were comparable to
those in wild-type mice. Surfactant pool sizes, surfactant synthesis,
and lung morphology were similar in SP-C (/) and SP-C (+/+)
mice. Lamellar bodies were present in SP-C (/) type II cells, and
tubular myelin was present in the alveolar lumen. Lung mechanics
studies demonstrated abnormalities in lung hysteresivity (a term used
to reflect the mechanical coupling between energy dissipative forces
and tissue-elastic properties) at low, positive-end, expiratory
pressures. The stability of captive bubbles with surfactant from the
SP-C (/) mice was decreased significantly, indicating that SP-C
plays a role in the stabilization of surfactant at low lung volumes, a
condition that may accompany respiratory distress syndrome in infants
and adults.
Introduction
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-helical structure with the -helix
capable of spanning a lipid bilayer. The hydrophobicity of SP-C is
augmented further by palmitoylation of two amino-terminal cysteine
residues located at positions 5 and 6 (5, 6). Together, these features
account for the strong association of SP-C with surfactant phospholipids.
Materials and Methods
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Materials and Methods
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References
) were bred to establish a colony of SP-C (+/+), SP-C (+/), and SP-C (/) mice. All mice were maintained in a pathogen-free
barrier containment facility with filtered air, water, and autoclaved food.
Analysis of RNA and Protein Expression.
Lung RNA was extracted from the lungs of adult SP-C (+/+), (+/),
and (/) mice by tissue homogenization in 4 M guanidinium isothiocyanate solution and centrifuge separation by using phase-lock gels as recommended (5' 
3'). Ten micrograms of total lung RNA from
each genotype was separated by agarose gel electrophoresis and
transferred to nylon membranes for hybridization analysis. Membranes
were probed with either murine SP-B or murine SP-C cDNA clones as
described (15). The relative abundance of mRNA species were quantitated
by using a PhosphorImager and IMAGEQUANT software (Molecular Dynamics). Analysis of the minor SP-C RNA species detected by Northern blot analysis was characterized further using by
exon-specific PCR analysis of cDNA generated by reverse transcription
(RT) of lung RNA. RT cDNA derived from lung RNA of SP-C (/) mice
was subject to 25 cycles of PCR amplification with an exon 1-specific 5' primer and an exon 4- or exon 5-specific 3' primer (5' exon 1 primer: TCT TGA TGG AGA CTC CAC CG; 3' exon 4 primer: CTG GCT TAT
AGG CCG CTA GG; 3' exon 5 primer: TCC GAT GCT CAT CTC AAG GA).
Detection of Alveolar Surfactant Proteins by Western Blot Analysis. Surfactant protein in alveolar lavage was separated by SDS/PAGE on 10-20% Tricine gels, and samples were normalized to 2 µg of saturated phosphatidylcholine (Sat PC) and loaded onto the gel for immunoblot analysis. The primary antibody was a rabbit antirecombinant SP-C serum that was used at a dilution of 1:25,000. The high-titer anti-SP-C antibody was raised against a modified human recombinant, 34-aa SP-C peptide (provided by Byk-Gulden Pharmazeutika). The specificity of the antibody for the mature form of SP-C was characterized (16). Western blot analysis of alveolar lavage proteins was performed with polyclonal antibodies to SP-A, SP-B, and SP-D as described (17).
Lung Morphology and Ultrastructure.
Lung morphology was visualized by conventional histological staining of
paraffin-embedded tissue, and type II cells were visualized by
immunostaining with an antibody specific to SP-B (15). Lungs from three
SP-C (/) and SP-C (+/+) 8-week-old mice were collected after
inflation fixation and prepared for electron microscopy as described
(17).
Sat PC Pool Sizes and Precursor Incorporation into Sat PC. The amount of Sat PC in lipid extracts of alveolar lavage and lung tissue after alveolar lavage from 8-week-old mice (n = 8 for each group) was determined as described (4), as was the incorporation rate of [3H]choline into Sat PC.
Surface Activity of Surfactant. Large aggregate surfactant was isolated from alveolar lavages by centrifugation at 40,000 × g for 15 min over 0.8 M sucrose in 0.9% NaCl cushion. The large aggregate surfactant was recovered from the sucrose interface. The surface activity of three pools (three mice per pool) of isolated large aggregate for each genotype group was measured with a captive bubble surfactometer. The concentration of each sample was adjusted to 3 nmol Sat PC/µl in 0.9% NaCl, and 3 µl of the surfactant was applied to the air-water interface of a 23.1 ± 0.9 µl bubble at 37°C. The surface tension was measured every 10 sec, equilibrium surface tension was measured at 60 sec, and bubble pulsation was started (4). The minimum surface tensions after 65% volume reduction of the bubbles were measured for the fifth pulsation. To test the ability of isolated surfactant to stabilize small bubbles, 3 µl of 0.3 nmol Sat PC/µl large aggregate surfactant was applied to the air-water interface and the bubble was adjusted to a 1.3 ± 0.1 µl volume; surface tension was measured for 12 min. From the calculation of bubble surface areas, it was determined that 10 times less Sat PC would maintain a similar surfactant concentration to surface area for the smaller-size bubble.
Lung Mechanics.
For lung mechanics studies, mice were anesthetized with 0.1 ml/10 g
of a mixture containing xylazine (2.0 mg/ml) and ketamine (40 mg/ml). Two-thirds of the dose was given to induce anesthesia, with
the remaining given when the animals were attached to the ventilator. A
tracheostomy was performed and a polyethylene cannula (1.0 cm, i.d. = 0.023 cm) was inserted. Mice were ventilated with a tidal volume of 8 ml/kg at a rate of 450 breaths per minute, with a positive
end-expiratory pressure (PEEP) of 0, 1, 2, and 4 cm
H2O, using a custom-designed ventilator
(flexiVent; Scireq, Montreal). This ventilator allowed us to measure
lung function by using a modification of the low-frequency forced
oscillation technique. Respiratory input impedance was measured between
0.25 and 20 Hz by applying a composite signal containing 19 mutually prime sinusoidal waves during pauses in regular ventilation. For each
measurement, the ventilator was paused for 1 sec with the mouse exposed
to the desired PEEP level, ensuring the appropriate end-expiratory
pressure. The expiratory valve then was closed and the
oscillatory-forcing function was applied by the piston. As the mouse
was exposed to a closed system during the measurements, changes in lung
volume were avoided. The constant-phase model described by Hantos
et al. (18) was used to partition respiratory input
impedance into components representing the mechanical properties of the
airway resistance and a constant-phase tissue component (Gtissue-Htissue)/w
, where
Gtissue and Htissue are coefficients for tissue resistance and tissue
elastance, w is the angular frequency, and determines the frequency dependence of the real and imaginary parts of the impedance. Hysteresivity describes the mechanical coupling between tissue resistance, and elastance and is calculated as
Hysteresivity = Gtissue/Htissue. The calibration procedure
removes the impedance of the equipment and tracheal tube. The results
reported represent the mechanical properties of the mice alone.
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SP-C Null Mutant Mice.
Five ES colonies were identified that carried the correctly targeted
SP-C allele (Fig. 1 A and
B). Three of the ES clones were microinjected into mouse
blastocysts that produced 34 chimeric mice. Chimeric males were bred to
NIH Swiss black mice. Germ-line transmission to F1 offspring was
identified by Southern blot analysis of BglII-digested DNA
by using the same 5' probe with which the 5.7-kb BglII band
increased to 7.4 kb. Germ-line transmission of the modified SP-C allele
was detected in offspring of chimeric mice generated from each of the
three injected ES cell clones. F1 offspring heterozygous for the
targeted SP-C allele were bred to produce SP-C (+/+), (+/), and
(/) mice (Fig. 1C). The SP-C (/) mice were
viable at birth and were similar to normal littermates in growth and
reproduction. SP-C (/) mice have been maintained for eight
generations in the vivarium.
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SP-B and SP-C mRNA and Protein Levels.
SP-C and SP-B mRNA was assessed by Northern blot analysis of lung RNA
from SP-C (+/+), (+/), and (/) mice. SP-C mRNA levels in
lungs from SP-C (+/) mice were approximately half the level (63%)
of SP-C (+/+) mice. No mature SP-C mRNA was detected in SP-C
(/) mice (Fig. 2A). A
faint band of hybridization smaller than the SP-C mRNA was detected in
lungs from SP-C (/) mice. This mRNA species was present at 
1%
of the wild-type SP-C mRNA. Lung RNA was converted to cDNA for RT-PCR
analysis to determine the size and sequence of the minor band. RT-PCR
analysis of SP-C (/) lung RNA by using an exon 1 primer coupled
with exon 4 or 5 downstream primers produced single bands that were
consistent with an SP-C cDNA product wherein exon 2 was absent.
Sequence of the PCR products confirmed SP-C sequence that was a direct splice from the end of exon 1 to the start of exon 3 (data not shown).
Thus, the minor SP-C mRNA species found by Northern blot in SP-C
(/) mice is an alternate splice eliminating exon 2 and the
encoded mature airway peptide. No band corresponding to the full-length
SP-C product was detected. SP-B mRNA expression was unaltered in SP-C
(+/) or SP-C (/) mice (Fig. 2A).
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/) mice, whereas the
mature SP-C peptide was readily detected by Western blot of alveolar
lavage from wild-type SP-C (+/+) and SP-C (+/) mice (Fig.
3A). Densitometric
quantitation of the SP-C (+/+) and (+/) bands indicated
that in the (+/) mice, SP-C peptide was present at 60% of the SP-C
level in SP-C (+/+) mice. This result was consistent with SP-C
expression from a single allele in the (+/) mice. The mature form
of the SP-B protein was present in alveolar lavage of SP-C (/)
mice equivalent to SP-B levels in SP-C (+/+) mice (Fig.
3B). The concentrations of surfactant proteins SP-A and SP-D
showed modest differences (Fig. 3B). Quantitative values for
SP-B in SP-C (/) mice were 0.89 ± 0.25 relative to SP-C (+/+) mice, with SP-B levels set to a valve of 1 ± 0.36. The
SP-A value in SP-C (/) mice was 1.34 ± 0.18 of SP-C (+/+)
mice, and the SP-D value in SP-C (/) mice was 0.84 ± 0.44 of the SP-D in SP-C (+/+) mice. Collectively, the Western
blot analysis for SP-A, SP-B, and SP-D indicates that concentrations of
these surfactant proteins were not influenced by SP-C and that these
surfactant proteins are regulated independent of SP-C.
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Lung Morphology.
Morphology of lung tissue from SP-C (/) mice was
indistinguishable from the SP-C (+/) or wild-type mice. Alveolar
and bronchiolar structures were normal with no indications of
inflammation. The number of type II cells appeared equivalent in SP-C
(/) and (+/+) mice (data not shown). The lungs of SP-C
(/), (+/), and (+/+) mice were examined by electron
microscopy to determine whether the elimination of SP-C altered type II
cell morphology or intra- and extracellular forms of pulmonary
surfactant. In adult SP-C (/) mice, numerous mature well
organized lamellar bodies (the intracellular form of surfactant) were
detected in type II cells of both SP-C (/) and SP-C (+/+) mice
(Fig. 4 A and B).
Fusion of multivesicular bodies to form lamellar bodies was observed in
SP-C (/) mice. Extracellular tubular myelin forms of pulmonary surfactant were readily detected in micrographs of the alveoli from
SP-C (/), (+/), and (+/+) mice (Fig. 4 C and
D). The size and abundance of lamellar bodies were assessed
by electron microscopy and were unaltered (data not shown).
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Sat PC Pool Sizes and Precursor Incorporation into Sat PC.
Surfactant Sat PC pool size in alveolar lavage and total lung (alveolar
lavage plus lung tissue after lavage) were similar in SP-C (/)
and SP-C (+/+) mice. The alveolar Sat PC was 10.7 ± 0.5 µmol/kg for SP-C (/) mice and 9.7 ± 0.5 µmol/kg for
SP-C (+/+) mice. Total lung Sat PC was 31.9 ± 1.4 µmol/kg
for SP-C (/) mice and 31.2 ± 1.1 µmol/kg for SP-C
(+/+) mice. [3H]Choline incorporation into
alveolar lavage Sat PC of SP-C (/) mice was 615 ± 49 cpm
and 681 ± 49 cpm for SP-C (+/+) mice. Incorporation into total
lung Sat PC was 2,575 ± 157 cpm for SP-C (/) mice and
2,314 ± 169 cpm for SP-C (+/+) mice. The secretion rate of Sat
PC was 21.4 ± 1.3% in SP-C (/) mice and 25.2 ± 1.5%
in SP-C (+/+) mice, and no significant differences were detected
between the groups. Thus, lack of SP-C does not alter surfactant
synthesis rate, secretion rate, or the surfactant pool size.
Surface Activity of Surfactant and Lung Mechanics.
Measurements made at increasing end-expiratory pressures from 0 to 4 cm
H2O showed no significant changes in airway
resistance, Gtissue, and Htissue between SP-C (+/+) and SP-C
(/) mice. However, statistically significant lower hysteresivity
was seen in all SP-C (/) mice at each end-expiratory pressure,
indicating reduced viscoelasticity of the lungs of SP-C (/) mice
(Fig. 5).
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/) mice was normal and showed equilibrium surface tension
and minimum surface tension similar to that from SP-C (+/+) mice
(Fig. 6A). The difference in
altered hysteresivity was considered to reflect altered surfactant
activity at low-end expiration or small alveolar volumes. Therefore,
the surface tension of surfactant from SP-C (+/+) and (/) mice
was evaluated in vitro by using a minimal-size bubble (1.3 µl) in the captive-bubble surfactometer to assess surfactant activity
in bubbles with small radii. Stability of small bubbles was decreased
markedly with the SP-C (/) surfactant, whereas equilibrium
surface tension was unaltered with surfactant from SP-C (+/+) mice
(n = 4 measurements). The changes in surface tension
are shown in Fig. 6B. The results are consistent with a role
for SP-C in stabilization of phospholipid packing at small bubble
radius and facilitation of the recruitment of phospholipids from the
subphase to the surface film.
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The mouse SP-C gene was inactivated by gene targeting. The lungs
of the SP-C (/) mice lack detectable mature SP-C or precursor SP-C (proSP-C). In the absence of detectable SP-C, these mice survive
and grow normally with no adverse effects on health, reproduction, or
pulmonary function. SP-B mRNA levels and the level of SP-A, SP-B, and
SP-D proteins were unaltered in the alveolar lavage of SP-C (/)
mice. Both SP-C and SP-B are hydrophobic peptides that dramatically
enhance surface activity of surfactant phospholipids. The survival of
the SP-C (/) mice in the absence of the mature active SP-C
peptide indicates that SP-C is not required for the surface properties
of the alveolar phospholipid films required for respiratory function
in vivo. However, subtle abnormalities in lung mechanics and
the instability of SP-C (/) surfactant at low bubble size support
the importance of SP-C in surfactant function at low lung volumes such
as that seen in respiratory distress syndrome in adults and preterm
infants. The present findings in the SP-C (/) mice are distinct
from results reported for SP-B gene-targeted mice and infants with
hereditary SP-B deficiency, wherein severe respiratory failure occurs
at birth (15, 19). In the SP-B (/) mice and human infants with
hereditary SP-B deficiency, the mature form of SP-C was absent or
diminished and an aberrant form of proSP-C was detected.
Histological analysis of lungs from SP-C (/) mice demonstrated
normal morphogenesis of the conducting and peripheral airways. Alveolar
structure was unaltered with a normal distribution of type II cells
(SP-B-positive) throughout the parenchyma with normal surfactant lipid
pool sizes in SP-C (/) mice. Radiolabeled choline incorporation
into Sat PC by lungs of SP-C (/) mice was similar to that in SP-C
(+/+) mice, indicating that SP-C is not essential for the synthesis
or secretion of pulmonary surfactant.
Lamellar bodies and tubular myelin, the unique intracellular and
extracellular forms of pulmonary surfactant, respectively, were
unaltered in SP-C (/) mice. In contrast, alterations in intra-
and extracellular forms of surfactant were detected when SP-A, SP-B,
and SP-D genes were targeted (17, 15, 20, 21). Pulmonary surfactant is
synthesized in type II cells and routed to multivesicular bodies, which
subsequently fuse to form the distinctive lamellar bodies that serve as
the presecretory storage form of surfactant. Upon secretion, lamellar
bodies form an extracellular reservoir of surfactant termed tubular
myelin. All of these structures were present in type II cells and air
spaces of SP-C (/) mice. This observation is distinct from those
in SP-B (/) mice, wherein enlarged multivesicular bodies were
observed in type II cells but no lamellar bodies were detected (15).
The aberrant lipid inclusions seen in SP-B (/) animals are
attributed in part to incorrect vesicle fusion, which is mediated by
SP-B. In SP-A (/) mice, normal lamellar bodies were detected
whereas the extracellular tubular myelin forms of surfactant were
virtually absent. SP-D (/) mice had an increased surfactant
phospholipid pool size and corresponding increase in the size of
lamellar bodies in type II cells (20, 21). The current findings
indicate that loss of SP-C is not required for formation of lamellar
bodies or tubular myelin structures.
SP-C has been hypothesized to play an important role in surfactant
function based on the ability of SP-C to enhance phospholipid surface
activity in vitro and to restore compliance in
surfactant-deficient immature rabbit and depleted animal lung models.
Instillation of SP-C preparations into the lungs of preterm rabbits and
lambs improved lung pressure volume curves and lung compliance (11). Similarly, SP-C-based synthetic surfactants improved lung compliance and oxygenation in adult rat and sheep lung injury models (12-14). When measurements of lung mechanics were evaluated at end expiration (low PEEP), the SP-C (/) mice had only modest increases in airway and tissue resistance, whereas hysteresivity was reduced significantly in these animals. A major function of surfactant is to reduce surface
tension in the alveoli as the lung volume decreases. This lowering of
surface tension at low lung volumes prevents airway collapse at end
expiration. Surfactant function is perhaps less important at high lung
volumes, where the collagen and elastin fiber network have more
influence on limiting lung inflation. The present finding,
demonstrating decreased hysteresivity at low PEEP pressures, suggests
that SP-C may stabilize phospholipid films at reduced lung volumes.
Phospholipid mixtures supplemented with purified SP-C or rSP-C or SP-B
were more effective in restoring lung compliance of premature rabbit
pups when ventilated with a PEEP of 4 cm H2O, similar to the conditions used in the analysis of the hysteresivity of
lungs from SP-C (/) mice (22). Surfactant films lacking SP-C were
unstable in small, captive bubbles compared with the stability of
surfactant preparations containing SP-C. This difference in stability
was detected only in bubbles with small radii wherein phospholipid
packing is high. Ultrastructural analysis of surfactant films in
vitro demonstrates the presence of both monolayers and multilayers
of phospholipids, the latter being evident when surfactant films are
highly compressed (23). Analysis of the alveolar surfactant film
structure by electron microscopy demonstrates that the surface film
contains regions of multilamellar stacks in vivo that are similar to those found in vitro (24). We hypothesize that
SP-C may stabilize adjacent lipid layers that form during film
compression and promote formation of surfactant reservoirs that
maintain availability of phospholipids for surface tension reduction at
the air-liquid interface. Both SP-C and SP-B enhance the rate of
surfactant film formation as phospholipids move onto an expanding
surface. However, SP-C-deficient surfactant is uniquely unstable at low
bubble volumes that also have a high angle of curvature, perhaps
leading to the instability of films seen in the captive-bubble assay.
These biophysical properties of SP-C-deficient surfactant are also
consistent with the loss of hysteresivity at low lung volumes seen
during forced oscillatory ventilation of SP-C (/) mice. Present
findings in SP-C (/) mice are consistent with previous work
demonstrating that SP-C (i) enhances the rate of film
formation, perhaps by contributing to regional differences in
phospholipid packing in membranes, and (ii) partitions into
and perhaps stabilizes densely packed gel phase phospholipids, such as
dipalmitoyl-phosphatidylcholine (25, 26). Despite the changes in lung
mechanics and surfactant activity, pulmonary function is remarkably
normal in SP-C gene-targeted mice.
The instability of SP-C-deficient surfactant and decreased
hysteresivity at low lung volumes predicts that under physiologic stress, SP-C deficiency may lead to alveolar instability that may
contribute to the pathogenesis of respiratory distress syndromes. SP-C
gene expression is inhibited after infection and exposure to tumor
necrosis factor and is decreased in lungs of infants with
respiratory distress syndrome, pneumonia, and pulmonary edema (27, 28).
Although SP-C is not required for normal pulmonary mechanics at normal
lung volumes, decreased surfactant proteins or phospholipids caused by
injury or infection may cause surfactant dysfunction, leading to
alveolar instability or acute alveolar collapse as seen in RDS, adult
respiratory distress syndrome, or pneumonia. Indeed, recent studies
identified subsets of children with selective deficiency of SP-C that
developed severe acute (29) and chronic lung disease in infancy
(R. S. Amin, S. E. Wert, R. P. Baughman, J. F. Tomashefski, L. M. Nogee, A. S. Brody, W. M. Hull, and
J.A.W., unpublished results; ref. 30).
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Acknowledgements |
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We acknowledge the efforts of Dr. Karen Yager and Sandy Falcone of the Transgenic Core Facility of the Children's Hospital Research Foundation for the blastocyst injections, Drs. John Baatz and Timothy Weaver for helpful discussions, and Ms. Ann Maher for assistance in manuscript preparation. This work was supported by National Heart, Lung, and Blood Institute Grants HL50046 (to S.W.G.), PPG HL61646 (to S.W.G., T.R.K., M.I., and J.A.W.), and HL38865 (to J.A.W.).
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Abbreviations |
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SP-C, surfactant protein C; RDS, respiratory distress syndrome; ES, embryonic stem; RT, reverse transcription; Sat PC, saturated phosphatidylcholine; PEEP, positive end-expiratory pressure.
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Footnotes |
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To whom reprint requests should be addressed at:
Children's Hospital Medical Center, Division of Pulmonary Biology,
3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail:
glass0{at}chmcc.org.
This paper was submitted directly (Track II) to the PNAS office.
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