Roberta Bruni

Protein Composition of Synthetic Surfactant Affects Gas Exchange in Surfactant-Deficient Rats

Synthetic surfactant peptides offer an opportunity to standardize the protein composition of surfactant. We tested the effect of phospholipids (PL) with synthetic full-length SP-B1-78 (B), mutant B (Bser), KL4 peptide (UCLA-KL4), and palmitoylated SP-C1-35 (C) on oxygenation and lung function in a surfactant-deficient rat model. Sixty-four adult rats were ventilated with 100% oxygen, a tidal volume of 7.5 mL/kg, and a rate of 60/min. Their lungs were lavaged with saline until the arterial PO2 dropped below 80 torr, when 100 mg/kg surfactant was instilled. Surfactant preparations included: PL (PL surfactant), PL + 3% B (B surfactant), PL + 3% B and 1% C (BC surfactant), PL + 3% UCLA-KL4(KL4 surfactant), PL + 3% Bser (Bser surfactant), and PL + 3% B and 1% UCLA-KL4 (BKL4 surfactant). Sixty minutes after surfactant instillation, positive end-expiratory pressure was applied for 5 min, and pressure-volume curves were determined in situ. The six surfactant preparations had a minimum surface tensions <10 mN/m on a Langmuir/Wilhelmy balance. Instillation of PL, Bser, and BKL4 surfactant increased mean arterial/alveolar PO2 (aADO2) ratios by 50-100% over postlavage values, whereas KL4 surfactant increased aADO2 ratios by 118%, B surfactant by 191%, and BC surfactant by 225%. Lung volumes at 30 cm H2O pressure were highest after treatment with BC surfactant, intermediate after B and KL4 surfactants, and lowest after BKL4, Bser, and PL surfactants. These data suggest that a surfactant preparation with a combination of synthetic B and C peptides surpasses synthetic B and KL4 surfactants in improving oxygenation and lung compliance in surfactant-deficient rats.

Inactivation of Surfactant in Rat Lungs

Although surfactant replacement therapy has dramatically improved the outcome of premature infants with respiratory distress syndrome, approximately 30% of treated infants show a transient or no response. Nonresponse to surfactant replacement therapy may be due to extreme lung immaturity and possibly surfactant inactivation. Surfactant inactivation involves aspecific biophysical events, such as interference with the formation or activity of an alveolar monolayer, and specific interactions with serum proteins, including antibodies, leaking into the alveolar space. As formulations containing surfactant proteins appear to better tolerate serum inactivation, we used an excised rat lung model to compare the susceptibility to serum inactivation of a mixture of synthetic phospholipids selected from surfactant lipid constituents, Exosurf (a protein-free synthetic surfactant), Survanta[containing surfactant proteins B and C (SP-B and -C)], and a porcine surfactant (containing SP-A, -B, and -C). For each of these preparations, we used pressure/volume determinations as an in situ measure of surfactant activity and retested the same preparations after mixing with human serum, a nonspecific surfactant inactivator. Human serum inactivated porcine surfactant to a lesser extent than Survanta, Exosurf, or synthetic phospholipids. Temperature exerted a significant effect on deflation stability, as shown by a greater lung compliance in untreated, normal lungs and a larger improvement in compliance after treating lavaged lungs with synthetic phospholipids at 37°C than at 22°C. We conclude that surfactant containing SP-A, -B, and -C is only moderately susceptible to inactivation with whole serum and may therefore exert a greater clinical response than protein-free surfactants or those containing only SP-B and-C.

A synthetic segment of surfactant protein A: structure, in vitro surface activity, and in vivo efficacy.

Surfactant protein A (SP-A) is a 248-residue, water-soluble, lipid-associating protein found in lung surfactant. Analysis of the amino acid sequence using the Eisenberg hydrophobic moment algorithm predicts that the SP-A segment spanning residues 114-144 has high hydrophobic moments, typical of lipid-associating amphipathic domains. The secondary structure, in vitro surface activity and in vivo lung activity of this SP-A sequence were studied with a 31-residue synthetic peptide analog(A114-144). Analysis of the secondary structure using circular dichroism and Fourier transform infrared spectroscopy indicated association with lipid dispersions and a dominant helical content. Surface activity measurements of A114-144 with surfactant lipid dispersions and the hydrophobic surfactant proteins B and C (SP-B/C) showed that A114-144 enhances surface activity under conditions of dynamic compression and respreading on a Langmuir/Wilhelmy surface balance. Synthetic surfactant dispersions containing A114-144 improved lung compliance in spontaneously breathing, 28-d premature rabbits to a greater degree than surfactant dispersions with synthetic SP-B/C and synthetic surfactant lipids alone. These observations indicate that inclusion of A114-144 may improve synthetic preparations currently used for surfactant replacement therapy.

Postnatal transformations of alveolar surfactant in the rabbit: changes in pool size, pool morphology and isoforms of the 32-38 kDa apolipoprotein.

To clarify perinatal transformations of surfactant we performed lung lavage in term fetuses and in 0-24-h-old newborn rabbits. Lavage fluid was separated into three pools, namely lavage pellet, lavage supernatant and cells. We found that at birth the pellet contains 94.1 +/- 1.4% (S.E.) saturated phosphatidylcholine, while the supernatant and cells contain traces of it. At birth the pellet contains secreted lamellar bodies while the supernatant lacks any recognizable structure. After birth, the alveolar saturated phosphatidylcholine level increases 5.1-times in 24 h, the proportions between pools reaching adult values in 90 min (pellet = 75.9 + 4.8%, supernatant = 22.7 +/- 4.9%), and small vesicles appear in the supernatant, probably originating from the turnover of alveolar surfactant during breathing. The saturated phosphatidylcholine associated with cells remains unchanged. At birth, the 32-38 kDa surfactant apolipoprotein appears to be less extensively sialylated than in adult life.

The life cycle of a low-molecular-weight protein of surfactant (SP-C) in 3-day-old rabbits

To clarify the metabolic cycle of a low-molecular-weight protein of surfactant (SP-C), we obtained alveolar surfactant from 3 day old rabbits killed 24 h after the tracheal administration of 32P or L-[35S]methionine (donors). Aliquots of this naturally labelled surfactant were administered into trachea to 3-day-old rabbits (recipients) which were killed after 1 min or 3, 8 or 24 h. We then analyzed the radioactivity associated with SP-C and with saturated phosphatidylcholine in fractions of lung lavage fluid and in lung homogenate. We found that alveolar SP-C is turned over faster than saturated phosphatidylcholine, that alveolar macrophages do participate in the removal of SP-C and that SP-C does not enter the fraction of alveolar surfactant that remains unsedimented after ultracentrifugation. Considering the whole lung, SP-C and saturated phosphatidylcholine are turned over at a comparable speed.

In chyloptysis, SP-A affects the clearance of serum lipoproteins entering the airways

Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chyloptysis, we noticed that surfactant could not be separated from contaminating serum lipoproteins and speculated that lipoproteins might interact with surfactant components. To clarify this point we immobilized surfactant protein (SP) A on microtiter wells and incubated it with 125I-labeled very low density lipoproteins (VLDLs), low-density lipoproteins, and high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in greater detail the interaction of SP-A with VLDLs, we found that the binding is time and concentration dependent; is inhibited by unlabeled lipoproteins, phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected by methyl α-d-mannopyranoside. Whole surfactant is a potent inhibitor of binding. Furthermore, we found that SP-A increases the degradation of VLDLs by alveolar macrophages and favors the association of VLDLs with alveolar surfactant. We conclude that SP-A influences the disposal of serum lipoproteins entering the airways and speculate that binding to alveolar surfactant might represent an important step in the interaction between exogenous substances and the lung.Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chyloptysis, we noticed that surfactant could not be separated from contaminating serum lipoproteins and speculated that lipoproteins might interact with surfactant components. To clarify this point we immobilized surfactant protein (SP) A on microtiter wells and incubated it with 125I-labeled very low density lipoproteins (VLDLs), low-density lipoproteins, and high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in greater detail the interaction of SP-A with VLDLs, we found that the binding is time and concentration dependent; is inhibited by unlabeled lipoproteins, phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected by methyl alpha-D-mannopyranoside. Whole surfactant is a potent inhibitor of binding. Furthermore, we found that SP-A increases the degradation of VLDLs by alveolar macrophages and favors the association of VLDLs with alveolar surfactant. We conclude that SP-A influences the disposal of serum lipoproteins entering the airways and speculate that binding to alveolar surfactant might represent an important step in the interaction between exogenous substances and the lung.

PARADOXICAL EFFECTS OF EXOGENOUS PROTEINS ON LUNG FUNCTION IN SURFACTANT-DEFICIENT RATS

Leakage of plasma proteins into the alveolar space can inhibit pulmonary surfactant function and worsen respiratory failure in ventilated preterm infants. We tested the effect of intratracheal instillation of fetal calf serum (FCS) and fresh frozen plasma (FFP) on lung function in ventilated rats who were made surfactant-deficient by saline lavage. Post lavage, the rats were treated with air placebo, Survanta, FCS or FFP, air placebo+ FCS or FFP 1 hour post lavage, or Survanta+ FCS or FFP 1 hour post lavage. After 2 hours of ventilation, pressure volume curves were performed and the lungs relavaged. FCS instillation rapidly improved oxygenation when given immediately post lavage or 1 hour after placebo or Survanta instillation, whereas FFP instillation never improved oxygenation. FCS instillation increased post-treatment lavage phospholipid values, but FFP did not. Both FCS and FFP decreased lung volume, but the negative effect of FFP exceeded that of FCS. Surfactant aggregate sizing of the nal lung lavages by dynamic light scattering showed a definite shift towards smaller aggregates after FFP, but not after FCS, instillation. These data suggest that intratracheal instillation of FCS improves oxygenation and preserves the alveolar presence of phospholipids and large surfactant aggregates, whereas FFP decreases oxygenation and surfactant aggregate size in surfactant-deficient lavaged rats.Leakage of plasma proteins into the alveolar space can inhibit pulmonary surfactant function and worsen respiratory failure in ventilated preterm infants. We tested the effect of intratracheal instillation of fetal calf serum (FCS) and fresh frozen plasma (FFP) on lung function in ventilated rats who were made surfactant-deficient by saline lavage. Post lavage, the rats were treated with air placebo, Survanta, FCS or FFP, air placebo+ FCS or FFP 1 hour post lavage, or Survanta+ FCS or FFP 1 hour post lavage. After 2 hours of ventilation, pressure volume curves were performed and the lungs relavaged. FCS instillation rapidly improved oxygenation when given immediately post lavage or 1 hour after placebo or Survanta instillation, whereas FFP instillation never improved oxygenation. FCS instillation increased post-treatment lavage phospholipid values, but FFP did not. Both FCS and FFP decreased lung volume, but the negative effect of FFP exceeded that of FCS. Surfactant aggregate sizing of the nal lung lavag...

Effects of Mechanical Ventilation on Surfactant Aggregate Size. 1471

Surfactant aggregate size correlates with function in vivo. We have compared the effects of mechanical ventilation on the aggregate size of synthetic surfactants in the surfactant depleted rat lung model, lavaged before surfactant treatment and after 1 hour of ventilation (FiO2 = 1.0, rate = 60, VT =7.5 mL/Kg). Aggregate size was measured on a Mitech laser beam sample sizer. We used phospholipids and palmitic acid (PL) alone, or PL plus synthetic peptides: PL + 3% B1-78 (B), PL + 3% B1-78& 1% C1-35 (BC), PL + 3% KL4 (KL4), PL + 3%B1-78 & 1% KL4 (BKL4), and PL + 3% of a serine analog of B1-78(Bser) at 25 mg PL/ml, using a dose of 100 mg/kg body weight. PL and sham treatment were used as controls. Surfactants, initial and final wash were assessed for particle size, and total proteins were measured in each lavage. Synthetic surfactant vesicles had an apparent diameter (AD) of 4-6 μm or more that did not change after surface cycling for 3 hours. In all groups, the first wash contained large particles with an AD of 1-6 μm. In the second wash, the sham treated and the BKL4 groups contained primarily small vesicles with an AD less than 0.2 μm. B, KL4, and Bser treated rats had a significant fraction of vesicles with an AD of 1-4 μm. PL had vesicles with an AD of both less than 0.1 and 1-2 μm. Protein content increased 2-20 folds after ventilation, but the increase did not correlate with vesicle size. These data suggest that synthetic surfactant proteins in PL may undergo only partial large-to-small from conversion in an animal model of surfactant depletion during mechanical ventilation.

LOCALIZATION OF SYNTHETIC SURFACTANT PROTEIN SP-B IN PHOSPHOLIPID VESICLES.|[dagger]| 1947

Surfactant subtypes have been identified by gradient centrifugation and characterized by differences in protein composition, vesicle size, and surface activity. The presence of surfactant proteins in each subtype is thought to be critical to the formation of aggregates and their function (Baritussio, Am J Physiol 1994;266:L436). To evaluate whether synthetic surfactant peptides interact with lipids in a manner comparable to native surfactant proteins, we prepared surfactant vesicles containing synthetic 125I-SP-B1-78 and phospholipids (DPPC:PG:Palmitic acid, 7:2:1) [PL], with and without palmitoylated synthetic SP-C1-34. We separated the aggregates by continuous sucrose gradient (0.1-0.8 M; 100,000 g for 48 h), collecting 50 fractions, and analyzed the PL and peptide profile. The PL peak appeared in fraction 17 and > 80% of PL was recovered in fractions 15-17, at a density of 1.07 g/mL, irrespective of the presence of SP-C. In the SP-B/PL aggregates, SP-B peaked at 51% in fraction 15 and > 95% of SP-B appeared in fractions 11-17. In the SP-B&SP-C/PL aggregates, 17% of the SP-B was detected at the bottom of the gradient with <5% PL and ≈78% was recovered in fractions 5-15 with a peak of 28% in fraction 13. In both aggregates, small amounts of PL, but no SP-B, were recovered at the top of the gradient, at a density of about 1.010 g/mL. In summary, synthetic SP-B associates with phospholipid aggregates at a density of about 1.07 g/mL, as shown by the overlapping peaks. The presence of SP-C does not affect the phospholipid distribution, but changes the SP-B profile significantly. These data are consistent with those obtained for term rabbit surfactant and suggest that the insertion of synthetic surfactant proteins in a simplified phospholipid dispersion reproduces important characteristics of native surfactant.