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J Appl Physiol 104: 1101-1108, 2008. First published February 14, 2008; doi:10.1152/japplphysiol.00865.2007
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Surfactant proteins B and C are both necessary for alveolar stability at end expiration in premature rabbits with respiratory distress syndrome

Andreas Almlén,1 Guido Stichtenoth,1 Bim Linderholm,1 Marie Haegerstrand-Björkman,1 Bengt Robertson,1 Jan Johansson,2 and Tore Curstedt1

1Department of Molecular Medicine and Surgery, Karolinska Institutet, Karolinska University Hospital, Stockholm; and 2Department of Anatomy, Physiology and Biochemistry, Swedish University of Agricultural Sciences, Biomedical Centre, Uppsala, Sweden

Submitted 10 August 2007 ; accepted in final form 13 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Modified natural surfactant preparations, used for treatment of respiratory distress syndrome in premature infants, contain phospholipids and the hydrophobic surfactant protein (SP)-B and SP-C. Herein, the individual and combined effects of SP-B and SP-C were evaluated in premature rabbit fetuses treated with airway instillation of surfactant and ventilated without positive end-expiratory pressure. Artificial surfactant preparations composed of synthetic phospholipids mixed with either 2% (wt/wt) of porcine SP-B, SP-C, or a synthetic poly-Leu analog of SP-C (SP-C33) did not stabilize the alveoli at the end of expiration, as measured by low lung gas volumes of ~5 ml/kg after 30 min of ventilation. However, treatment with phospholipids containing both SP-B and SP-C/SP-C33 approximately doubled lung gas volumes. Doubling the SP-C33 content did not affect lung gas volumes. The tidal volumes were similar in all groups receiving surfactant. This shows that SP-B and SP-C exert different physiological effects, since both proteins are needed to establish alveolar stability at end expiration in this animal model of respiratory distress syndrome, and that an optimal synthetic surfactant probably requires the presence of mimics of both SP-B and SP-C.

pulmonary surfactant; respiratory distress syndrome; synthetic peptide

A MAJOR FUNCTION OF PULMONARY surfactant is to reduce surface tension, thus preventing the alveoli from collapsing at end expiration (39). Surfactant has a complex composition with at least 50 different lipids (6) and four specific proteins: surfactant protein (SP)-A, SP-B, SP-C, and SP-D (39). The predominant lipid component, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), is responsible for reducing surface tension to low values during expiration. The hydrophobic proteins SP-B and SP-C are critical for the adsorption of the surfactant film at the alveolar air-liquid interface (12, 39), while the hydrophilic proteins SP-A and SP-D are mainly involved in innate host defense (40).

A major cause of morbidity in preterm babies is primary surfactant deficiency with alveolar collapse. This may lead to mechanical disruption of the airway epithelium and impairment of lung mechanics and gas exchange, thus causing respiratory distress syndrome (RDS). Today, airway instillation of surfactant is in general use for treatment of RDS in preterm babies. The most satisfactory results have been obtained with preparations purified from animal lungs (17). Common components in these preparations are phospholipids, SP-B, and SP-C. SP-C is a very hydrophobic lipoprotein, which, in its longest form, has 35 amino acid residues and thioester-linked palmitoyl groups in the NH2-terminal part (11). The {alpha}-helical part of the molecule is inserted in the lipid bilayer and orientated near parallel to the phospholipid acyl groups (33). SP-B has 79 amino acid residues with 7 Cys residues, forming 3 intramolecular disulfide bridges. The remaining Cys is linked to the corresponding residue of another SP-B monomer (19). The homodimeric molecule may interact with the surface of the lipid bilayers by means of four or five amphipatic {alpha}-helices in each monomer (3). The importance of the hydrophobic proteins is illustrated by experimental and clinical observations (1, 34). Both SP-B and SP-C knockout mice are unable to establish normal breathing (15, 37). Infants with SP-B deficiency develop progressive, lethal respiratory failure in the neonatal period, while patients with SP-C deficiency will develop progressive lung fibrosis (26). Despite their great impact on adsorption of phospholipids at the air-liquid interface, the exact mechanisms of action of SP-B and SP-C are incompletely understood.

Surfactant preparations derived from animal lungs are expensive and have a limited supply, and, therefore, there is a need for synthetic surfactant substitutes that can be produced in large quantities for a reasonable cost. The physiological effects of synthetic surfactant preparations based on simple phospholipid mixtures and one peptide have been assessed in both premature animals with surfactant deficiency (13, 14, 18, 20, 22–24, 28) and in animals with acute RDS (5, 16, 31, 35, 36). In contrast to natural surfactant preparations, synthetic surfactants seem to need positive end-expiratory pressure (PEEP) (13, 20, 23), indicating that natural surfactant preparations are more effective in stabilizing the airways. The reasons for this difference are not yet understood, but may be due to the lipid composition as well as the amount, structure, and composition of the peptides.

Our laboratory has used synthetic surfactants based on SP-C33, which is an SP-C analog (2, 20). This peptide differs from human SP-C by removal of the two NH2-terminal amino acid residues, replacement of the palmitoylCys residues at positions 5 and 6 (positions 3 and 4 in SP-C33) with Ser, replacement of Leu14 with Lys, and replacement of the poly-Val part (positions 15–21 and 23–28) with a poly-Leu sequence (2, 20). Premature newborn rabbits with surfactant deficiency, treated with synthetic surfactant containing 2% of SP-C33 in DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (68:31 wt/wt) and ventilated with a standardized sequence of pressure without PEEP, have similar tidal volumes as those treated with a modified porcine surfactant preparation (Curosurf). However, lung gas volumes, reflecting the functional residual capacity, are much lower in animals treated with the synthetic surfactant (20). We have used this animal model to evaluate whether addition of native SP-B to phospholipids with or without SP-C/SP-C analog will increase alveolar stability.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
Phospholipids

DPPC, 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC), and POPG were obtained from Chiesi Farmaceutici, and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (PLPE) from Sigma-Aldrich.

Peptides

A mixture of phospholipids and the hydrophic proteins SP-B and SP-C were isolated from porcine lungs, as described previously (12). The proteins were separated from the phospholipids on a Sephadex LH-20 column in chloroform-methanol (2:1 vol/vol) (27) and mixed with synthetic phospholipids. For separation of SP-B from SP-C, size-exclusion chromatography on a Sephadex LH-60 column in chloroform-methanol-0.1 M HCl (19:19:2 by volume) was used (12). The contents of SP-B and SP-C were determined using amino acid analysis (32).

The peptide SP-C33 (IPSSPVHLKRLKLLLLLLLLILLLILGALLMGL) was provided by Chiesi Farmaceutici (Parma, Italy).

Surfactant Preparations

DPPC/PLPC/POPG/PLPE (55:25:15:5 by weight) or DPPC/POPG (68:31 wt/wt) dissolved in chloroform-methanol (1:1 vol/vol) were mixed with peptides in the same solvent. The peptide contents were 2% SP-C33, 4% SP-C33, 2% SP-B, 2% SP-C, 2% SP-C33 + 2% SP-B, 1% SP-C + 1.5% SP-B, or 2% SP-C + 3% SP-B. The mixtures were evaporated and resuspended in saline by gentle rotation in water (35°C) to a final phospholipid concentration of 80 mg/ml. The preparations were stored at 4°C.

Surface Activity Measurements

Two milliliters of the surfactant preparation, diluted with saline to 10 mg/ml, were added to a 15-ml (10 x 1.4 cm) capped glass tube that was rotated at 20 rpm for 7 days at 37°C to obtain a maximal change of the surface area. Aliquots were taken from the samples at time 0, and after 1, 3, and 7 days of cycling. Surface activity of the aliquots was measured in duplicates or triplicates using a captive bubble surfactometer (CBS) (4), and the results are presented as mean values. The test chamber was initially filled with 10% sucrose in saline. Approximately 2 µl of surfactant were injected into the sample chamber and allowed to migrate by buoyance to the agarose ceiling. An air bubble was then placed under the ceiling in contact with the surfactant preparation, and surface tension was measured from the time of bubble insertion. After 5 min of adsorption, the sample chamber was sealed, and the quasi-static cycling was initiated. The bubble was compressed stepwise until a surface tension <5 mN/m was reached or to 50% area compression and thereafter expanded to the initial size. This maneuver was repeated five times.

Stability of SP-C, SP-B, and SP-C33

Curosurf (containing SP-B and SP-C) and SP-C33 in DPPC/POPG 68:31 (wt/wt) were diluted to 5 mg/ml in 150 mM sodium chloride with 0.02% sodium azide to a final volume of 4.5 ml and incubated at 22°C with shaking. After 0, 7, and 14 days, the suspensions were centrifuged at 100,000 g for 30 min at 22°C, and the supernatants were removed. The pellets were dissolved in 1% (wt/vol) SDS and centrifuged again to remove denaturated and aggregated protein, and the resulting supernatants were lyophilized. The lyophilized supernatants were finally redissolved in sample buffer (12% glycerol, 2% SDS) and separated by SDS-PAGE on 10–16% Tris-Tricine gels under nonreducing conditions.

In Vivo Experiments

Preterm newborn rabbits obtained at a gestational age of 27 days (term 31 days), randomly allocated to different treatments, were tracheotomized at birth and received, via a tracheal cannula, one of the synthetic or semisynthetic preparations. Surfactant, 80 mg/ml, was administered at a dose of 2.5 ml/kg. Animals receiving the same dose of Curosurf served as positive controls, and nontreated littermates as negative controls. The animals were kept in plethysmograph boxes at 37°C and ventilated in parallel with 100% oxygen at a frequency of 40 breaths/min and an inspiration-to-expiration ratio of 1:1. To open up the lungs, peak inspiratory pressure was first set to 35 cmH2O for 1 min. Then pressure was lowered to 25 cmH2O for 15 min and further on to 20 cmH2O for 5 min and 15 cmH2O for 5 min. Finally, pressure was raised again to 25 cmH2O for 5 min (6, 12), after which the lungs were ventilated for an additional 5 min with nitrogen. The experiments were performed without PEEP. Tidal volumes were recorded every 5 min. At the end of the experiment, the tracheal cannula was clamped at end expiration, the trachea ligated, and the lungs were excised and weighed.

Determination of Lung Gas Volume

Lung gas volume, expressed as milliliters per kilogram body weight, was determined by water displacement technique (29) using the difference between the total volume of the lung (VLung) and the volume of the lung tissue (VTissue), divided by the body weight. VLung was derived by weighing the volume of water displaced by the lung submersed into a water bowl. VTissue was calculated by converting the wet weight of the lung into a volume by dividing this wet weight (WTissue) by the specific density of lung tissue. For this purpose, lung weight and VLung in 32 nonventilated newborn preterm rabbits (gestational age 27 days) was determined, and the specific density calculated to be 1.077 ± 0.03 (mean ± SD), giving VTissue (ml) = WTissue (g)/1.077.

The experiments were approved by the local ethical committee for animal research, Stockholms Norra Djurförsöksetiska Nämnd (205/04).

Lung Histology

The lungs were fixed by immersion in 4% neutral formalin, dehydrated, and embedded in paraffin. Transverse sections (thickness 3.5 µm) from the lower lobes, stained with hematoxylin and eosin, were examined by light microscopy, with particular reference to the alveolar expansion and desquamating airway epithelial cells. The proportion of well-aerated alveoli was estimated and classified semiquantitatively, according to a five-grade scale (0: 0, 1: 1–25, 2: 26–50, 3: 51–75, 4: >75%), while a four-grade score was used for estimation of desquamating airway epithelial cells (0 = absent, 1 = mild, 2 = moderate, 3 = prominent). These epithelial lesions have been described by light and electron microscopy in premature rabbits after a few minutes of ventilation (25). In addition, alveolar volume density was measured with a computer-aided image analyzer using total parenchyma as reference volume (7). The histological examinations were blinded, i.e., the examiner was unaware of the experimental conditions of individual animals.

Statistics

One-way ANOVA followed by Newman-Keuls multiple-comparison test was used for the in vivo data. Histological scores were analyzed by one-way ANOVA, nonparametric as described by Kruskal-Wallis, and followed by Dunn's multiple-comparison posttest. Statistical significance was considered when P < 0.05.

The data were statistically evaluated using GraphPad Prism 4.02 software (San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
The effects of different surfactant preparations were investigated in vitro in a CBS and in vivo by treatment of premature newborn rabbits. First, an unresolved mixture of SP-B and SP-C was added to DPPC/POPG 68:31 (wt/wt) to minimize possible modification, denaturation, or decomposition of proteins during isolation. Second, SP-B or SP-C was suspended in DPPC/PLPC/POPG/PLPE (55:25:15:5 by weight) to mimic the composition of natural surfactant phospholipids regarding disaturated species and phospholipid classes. Third, native SP-C was replaced with SP-C33 to evaluate the effect of SP-B in a synthetic mixture containing 2% SP-C33 in DPPC/PLPC/POPG/PLPE (55:25:15:5 by weight) or DPPC/POPG (68:31 wt/wt).

Purity of SP-B and SP-C Fractions

Three batches of SP-B and SP-C were prepared. In batch 1, SP-B and SP-C were not separated from each other, and their weight ratio was found to be 3:2 by amino acid analysis. The SP-B and SP-C fractions in batch 2, which were used in the experiments with DPPC/PLPC/POPG/PLPE, contained small amounts of the other surfactant protein. Thus the SP-B fraction contained 10% of SP-C, and the SP-C fraction contained 20% SP-B. In the SP-B fraction of batch 3, used in experiment with the phospholipid mixture DPPC/POPG, no SP-C could be detected by amino acid analysis.

In Vivo Activity

Treatment with semisynthetic surfactants containing unresolved native hydrophobic proteins and synthetic phospholipids.   Premature animals treated with low (1.5% SP-B + 1% SP-C) or high (3% SP-B + 2% SP-C) dose of proteins in DPPC/POPG 68:31 (wt/wt) had mean tidal volumes of ~24–25 ml/kg after 30 min of ventilation. Corresponding values for the groups treated with Curosurf or SP-C33 surfactant were similar, while nontreated controls had statistically lower tidal volumes (Table 1). Curosurf-treated animals had median lung gas volumes of ~21 ml/kg, which was significantly higher than that in animals treated with semisynthetic surfactants or SP-C33 surfactant (Fig. 1). The animals treated with the surfactant containing 3% SP-B + 2% SP-C had a slightly higher lung gas volume than animals treated with surfactant containing 1.5% SP-B + 1% SP-C, but the difference was not significant (Fig. 1). Furthermore, animals treated with semisynthetic surfactant as a group had significantly higher lung gas volumes than animals treated with SP-C33 surfactant (P < 0.05). The increase in lung gas volume is not an effect of peptide content, because preterm newborn rabbits treated with 2 or 4% SP-C33 in DPPC/POPG 68:31 (wt/wt) had similar lung gas volumes (Fig. 2). Increase of SP-C33 content from 2 to 4% had small but not statistically significant effects on mean tidal volumes of 10 and 14 ml/kg, respectively, after 30 min of ventilation, compared with the Curosurf group with 19 ml/kg (Table 1). Furthermore, the groups treated with 2 and 4% SP-C33 in DPPC/POPG had similar lung gas volumes, 4–5 ml/kg, whereas the Curosurf-treated animals had 11 ml/kg (Fig. 2). No statistically significant differences were found between groups treated with 2 or 4% SP-C33 regarding tidal volumes, lung gas volumes, and histological observations (Tables 1 and 2, Fig. 2).


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  		Table 1. Tidal volumes after 30 min of ventilation in preterm newborn rabbits treated with 200 mg/kg (80 mg/ml) of phospholipids mixed with peptides

 

Figure 1
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  		Fig. 1. Lung gas volumes in preterm newborn rabbits treated with unresolved mixtures of surfactant protein (SP)-B and SP-C in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) (68:31 wt/wt) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or in nontreated controls. The lines indicate median values. Levels of statistical significance: #P < 0.01–0.001 vs. all groups; *P < 0.01 vs. all surfactants; §P < 0.05 vs. all animals receiving surfactant containing the mixtures of SP-B and SP-C in DPPC/POPG.

 

Figure 2
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  		Fig. 2. Lung gas volumes in preterm newborn rabbits treated with 2 or 4% SP-C33 in DPPC/POPG (68:31 wt/wt) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or in nontreated controls. The lines indicate median values. Levels of statistical significance: ***P < 0.001 vs. all surfactants; ###P < 0.001 vs. 2 and 4% SP-C33.

 

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  		Table 2. Histological observations in preterm newborn rabbits treated with 2 or 4% SP-C33 in DPPC/POPG (68:31 wt/wt) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or nontreated controls

 
Effect of SP-B in synthetic phospholipids.   The experiments were performed to evaluate the effect of SP-B, SP-C, SP-C33, or combinations thereof in different phospholipid mixtures on tidal volumes, lung gas volumes, alveolar expansion, and desquamating airway epithelial cells.

PHOSPHOLIPID MIXTURE: DPPC/PLPC/POPG/PLPE.   Animals treated with Curosurf had, after 30 min of ventilation, a mean tidal volume of 23 ml/kg at a pressure of 25 cmH2O (Table 1). Corresponding values for the groups treated with synthetic preparations were 15–17 ml/kg. No statistical differences were observed between the groups. Nontreated animals had statistically lower tidal volumes (3.0 ml/kg) than the surfactant-treated groups.

Animals treated with Curosurf had a median lung gas volume of ~9 ml/kg, which was significantly higher than those obtained for animals treated with synthetic surfactant containing only one protein (Fig. 3). Animals treated with synthetic surfactants containing either SP-B or SP-C had a tendency to higher lung gas volume than those given surfactants with SP-C33, probably due to small amounts of the other protein in the purified fractions. Addition of SP-B to SP-C33 surfactant significantly increased lung gas volume to similar levels as those obtained for animals treated with Curosurf (Fig. 3). Furthermore, animals treated with surfactant containing both SP-B and SP-C33 had a higher grade of alveolar expansion than those treated with surfactant preparations containing SP-B or SP-C33. Similar results were obtained by alveolar volume density measurements (Table 3).


Figure 3
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  		Fig. 3. Lung gas volumes in preterm newborn rabbits treated with peptides in DPPC/1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG)/PLPE (55:25:15:5 by weight) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or in nontreated controls. The lines indicate median values. Levels of statistical significance: *P < 0.05–0.001 vs. SP-C, SP-C33, and SP-B; #P < 0.05–0.001 vs. all surfactants.

 

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  		Table 3. Histological observations in preterm newborn rabbits treated with peptides in DPPC/PLPC/POPG/PLPE (55:25:15:5 by weight) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or nontreated controls

 
PHOSPHOLIPID MIXTURE: DPPC/POPG.   Animals treated with Curosurf or surfactant containing both SP-C33 and SP-B had, after 30 min of ventilation, mean tidal volumes of ~10–13 ml/kg at 25 cmH2O, which is lower than in the previous experiments (Table 1), possibly due to a different degree of maturity of the preterm rabbits. Animals treated with surfactant containing SP-C33 or SP-B had a trend toward lower tidal volumes than those treated with surfactant containing both SP-C33 and SP-B, but the differences were not statistically significant (Table 1). Treatment with surfactant containing both SP-B and SP-C33 resulted in lung gas volumes of ~11 ml/kg, which is significantly higher than the 5 ml/kg observed for animals treated with synthetic surfactants containing either SP-B or SP-C33, but significantly lower than those obtained after treatment with Curosurf (Fig. 4). The results were confirmed by the histological examinations (Table 4, Fig. 5). Airway epithelial disruption was absent or very mild in animals treated with any type of surfactant and moderate in nontreated controls (Fig. 5). In animals treated with surfactant containing SP-B or SP-C33, alveolar air expansion was unsatisfactory, but improved after treatment with a synthetic surfactant containing both SP-B and SP-C33 or Curosurf (Table 4). Similar, but not statistically significant, results were obtained by alveolar volume density measurements.


Figure 4
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  		Fig. 4. Lung gas volumes in preterm newborn rabbits treated with peptides in DPPC/POPG (68:31 wt/wt) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or in nontreated controls. The lines indicate median values. Levels of statistical significance: ***P < 0.001 vs. SP-B + SP-C33 and Curosurf; ###P < 0.001 vs. all synthetic surfactants; §§P < 0.01 vs. SP-C33 and SP-B.

 

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  		Table 4. Histological observations in preterm newborn rabbits treated with peptides in DPPC/POPG (68:31 wt/wt) (80 mg/ml, 2.5 ml/kg), Curosurf (80 mg/ml, 2.5 ml/kg), or nontreated controls

 

Figure 5
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  		Fig. 5. Representative histological sections showing alveolar expansion in preterm newborn rabbits after administration of Curosurf (A), 2% SP-B (B), 2% SP-C33 (C), or 2% SP-B + 2% SP-C33 (D) in DPPC/POPG (68:31 wt/wt). E: nontreated animals served as controls and also showed moderate epithelial disruption (arrows). Hematoxylin and eosin, x10.

 
In Vitro Activity

Phospholipid mixture: DPPC/PLPC/POPG/PLPE.   During adsorption, surfactant mixtures containing SP-C or SP-C33 reached surface tension of ~25 mN/m within 1 s, while surfactant preparations containing SP-B, with or without SP-C, had a slightly slower adsorption. However, for all surfactant mixtures, rotation for 1 day at 37°C resulted in slower adsorption (Fig. 6). The compression needed to reach a surface tension of 5 mN/m also increased (Table 5).


Figure 6
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  		Fig. 6. In vitro surface activity of surfactant suspensions containing 10 mg/ml of DPPC/PLPC/POPG/PLPE (55:25:15:5 by weight) combined with 2% SP-C33 + 2% SP-B, 2% SP-C, 2% SP-C33, or 2% SP-B. The samples were rotated at 20 rpm at 37°C to obtain a maximal change of the surface area. Aliquots were analyzed before (0) and after 1, 3, and 7 days of rotation. Surface tension of the surfactant preparations was measured in the captive bubble surfactometer after 1, 10, and 300 s of adsorption for each sample. Two or three measurements were performed for each time point, and the results are expressed as median (range). Due to experimental problems, no data were recorded for the sample containing 2% SP-B after 7 days.

 

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  		Table 5. In vitro surface activity measured as compression needed to obtain surface tension of 5 mN/m in the captive bubble surfactometer after ≤7 days of cycling

 
Phospholipid mixture: DPPC/POPG.   Surfactant mixtures containing SP-C33, with or without SP-B, had a rapid adsorption with surface tension of ~25 mN/m after 1 s, while surfactant containing SP-B had a higher surface tension after 1 and 10 s. Preparations containing only one protein already had, after 1 day of rotation at 37°C, values >40 mN/m after 1 s of adsorption, while the preparation containing both SP-C33 and SP-B had values <30 mN/m, still after 7 days of rotation (Fig. 7). These values are in agreement with those obtained with the quasi-static cycling showing that <20% of compression is needed to obtain surface tension of 5 mN/m after 7 days of rotation for the surfactant preparation containing both SP-B and SP-C33 (Table 5).


Figure 7
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  		Fig. 7. In vitro surface activity of surfactant suspensions containing 10 mg/ml of DPPC/POPG (68:31 wt/wt) combined with 2% SP-C33 + 2% SP-B, 2% SP-C33, or 2% SP-B. For details, see legend to Fig. 6.

 
Stability of SP-C, SP-B, and SP-C33

Curosurf and SP-C33 in DPPC/POPG 68:31 (wt/wt) were incubated for up to 14 days. Analysis of nonaggregated protein showed essentially the same amounts of SP-B, SP-C, and SP-C33, respectively, at the different time points. This indicates that combining SP-B and SP-C does not confer increased stability of any of the proteins, relative to SP-C33 alone (C. Nerelius, J. Johansson, unpublished observations).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 
We show here that addition of SP-B to surfactant containing only SP-C, or the synthetic analog SP-C33, increases lung gas volumes at end expiration in ventilated preterm rabbits. This occurs without any effect on tidal volumes. SP-C isolated from lung tissue and the synthetic analog SP-C33, which contains poly-Val->poly-Leu substitutions to avoid loss of helical structure and formation of β-sheet aggregates (24), showed similar effects on tidal volumes and lung gas volumes. It is not known to what extent isolated SP-C or SP-C33 mimics native SP-C, but the equal effects of SP-C and SP-C33 alone suggest that their suboptimal performance is not caused by denaturation or chemical modifications, but rather by lack of SP-B.

SP-B and SP-C are the only known strictly hydrophobic proteins associated with lung surfactant. They constitute ~0.7 and 0.6% (wt/wt), respectively, of the modified surfactant preparation Curosurf (32), and ~1.3 and 4% (wt/wt), respectively, of natural surfactant (8). Numerous in vitro studies using surface balances and pulsating and captive bubbles have shown that SP-B or SP-C mixed with phospholipids show similar effects on surfactant adsorption, as well as minimum and maximum surface tension during cycling (see, e.g., Ref. 38). This has led to a common assumption that they perform similar and partly overlapping functions in alveolar surfactant. In contrast, studies using mice in which the genes for SP-B or SP-C have been individually knocked out have given a more complex picture. SP-B knockout mice, like humans with inherited deficiency of SP-B, develop fatal respiratory distress shortly after birth (9). The interpretation of these studies is in part complicated by the fact that SP-B knockout mice, like SP-B-deficient humans, lack mature SP-C and instead accumulate a late processing intermediate of pro-SP-C that has very low surface activity in mixture with phospholipids (21). The SP-B knockout mice are, therefore, practically devoid of both SP-B and SP-C. In addition to dysfunction of alveolar surfactant, SP-B deficiency results in severe alteration of the type II cell, including absence of normal lamellar bodies. This is probably underlying the lack of mature SP-C, since the final processing of pro-SP-C normally takes place in the lamellar body.

From the results showing increased lung gas volumes and the histological findings, we conclude that both SP-B and SP-C/SP-C33 are necessary for establishment of alveolar stability at end expiration in newborn premature rabbits ventilated without PEEP. In addition to the superior effects in premature newborn rabbits, the in vitro properties, as measured in the CBS, are better for surfactant containing both SP-B and SP-C/SP-C33. This applies to adsorption as well as minimum and maximum surface tension, and surface area reduction required to achieve low surface tension during cycling. Moreover, the surfactant containing both proteins appears to be more stable, as it stays active for up to 7 days, while the single protein mixtures lose surface activity already after 1–3 days. This is not likely to be caused by increased protein stability for the SP-B + SP-C mixtures compared with single proteins mixed with phospholipids, since gel electrophoresis showed nearly identical amounts of soluble proteins for up to 14 days.

Taken together, these data imply that SP-B and SP-C fulfill different functions in alveolar surfactant. This is not unexpected, considering that SP-B and SP-C are different from most structural aspects. The current findings indicate that exogenously delivered surfactant preparations require both SP-B and SP-C, or analogs thereof with similar functions, for optimal activity. This is in line with the behavior of SP-B-deficient mice and humans, but it is not anticipated from the behavior of SP-C-deficient mice. SP-C knockout mice have apparently normal respiration at birth, but develop signs of emphysema and interstitial pneumonitis over time, which suggests that SP-B alone is able to establish low alveolar surface tension and stable terminal air spaces at birth (15). The reasons for the discrepancies between the present findings and those from SP-C-deficient mice are not clear. It is possible that isolated porcine SP-B administered via the airways, together with a few synthetic phospholipid species, is less efficient than endogenous SP-B secreted into the alveoli, together with the native surfactant lipid mixture. SP-B is a complex molecule with several {alpha}-helices folded together by intra- and interchain disulfides (30), which makes it conceivable that partial loss of structure can take place during its isolation from surfactant phospholipids.

Attempts made during more than 20 yr to formulate a protein-containing synthetic surfactant preparation for treatment of respiratory distress have not yet resulted in a commercially available product. The synthetic surfactants studied so far have been based on synthetic or recombinant analogs of either SP-B or SP-C (10). All of these preparations show promising effects in animal models of lung disease, provided that a small PEEP is applied. The requirement of a PEEP is not considered to disqualify such synthetic surfactants from potential clinical use, since ventilation with PEEP is part of the routine management of patients with severe respiratory failure. However, modified natural surfactants show optimal activity also without PEEP, suggesting that they are functionally superior to synthetic surfactants based on single protein constituents. The present investigation shows that the presence of both SP-C, or an SP-C analog, and SP-B in a binary mixture of synthetic phospholipids is sufficient to establish high lung gas volumes in immature newborn rabbits, also in the absence of PEEP. However, none of the SP-B + SP-C/SP-C33 mixtures give as high lung gas volumes as modified natural surfactant Curosurf. The reasons for this are unknown, but may include changes in protein structure during isolation, differences between the synthetic analog SP-C33 and native SP-C, or that the synthetic mixture contains very few phospholipid species compared with natural pulmonary surfactant. The results presented herein suggest that determination of lung gas volumes can be used to identify an SP-B analog that, when combined with SP-C33, yields a synthetic surfactant that may be comparable to presently available modified natural surfactants for replacement therapy.


   DISCLOSURES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
ACKNOWLEDGMENTS
 REFERENCES
 
T. Curstedt, B. Robertson, and J. Johansson together have a $200,000 grant yearly from Chiesi Farmaceutici SpA. They also have coauthor patent applications regarding SP-C analogs with Chiesi Farmaceutici.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
REFERENCES
 
This work was supported by grants from the Swedish Research Council, Konung Oscar II Jubileumsfond, and Chiesi Farmaceutici.


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. Curstedt, Dept. of Molecular Medicine and Surgery, Section of Clinical Chemistry, Bldg. L2:03, Karolinska Univ. Hospital, SE-171 76 Stockholm, Sweden (e-mail: tore.curstedt{at}karolinska.se)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 ACKNOWLEDGMENTS
 REFERENCES
 

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