José M. Hernández-Juviel

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.

Critical Structural and Functional Roles for the N-Terminal Insertion Sequence in Surfactant Protein B Analogs

Background Surfactant protein B (SP-B; 79 residues) belongs to the saposin protein superfamily, and plays functional roles in lung surfactant. The disulfide cross-linked, N- and C-terminal domains of SP-B have been theoretically predicted to fold as charged, amphipathic helices, suggesting their participation in surfactant activities. Earlier structural studies with Mini-B, a disulfide-linked construct based on the N- and C-terminal regions of SP-B (i.e., ∼residues 8–25 and 63–78), confirmed that these neighboring domains are helical; moreover, Mini-B retains critical in vitro and in vivo surfactant functions of the native protein. Here, we perform similar analyses on a Super Mini-B construct that has native SP-B residues (1–7) attached to the N-terminus of Mini-B, to test whether the N-terminal sequence is also involved in surfactant activity. Methodology/Results FTIR spectra of Mini-B and Super Mini-B in either lipids or lipid-mimics indicated that these peptides share similar conformations, with primary α-helix and secondary β-sheet and loop-turns. Gel electrophoresis demonstrated that Super Mini-B was dimeric in SDS detergent-polyacrylamide, while Mini-B was monomeric. Surface plasmon resonance (SPR), predictive aggregation algorithms, and molecular dynamics (MD) and docking simulations further suggested a preliminary model for dimeric Super Mini-B, in which monomers self-associate to form a dimer peptide with a “saposin-like” fold. Similar to native SP-B, both Mini-B and Super Mini-B exhibit in vitro activity with spread films showing near-zero minimum surface tension during cycling using captive bubble surfactometry. In vivo, Super Mini-B demonstrates oxygenation and dynamic compliance that are greater than Mini-B and compare favorably to full-length SP-B. Conclusion Super Mini-B shows enhanced surfactant activity, probably due to the self-assembly of monomer peptide into dimer Super Mini-B that mimics the functions and putative structure of native SP-B.

Dynamic Surface Activity of a Fully Synthetic Phospholipase-Resistant Lipid/Peptide Lung Surfactant

Background This study examines the surface activity and resistance to phospholipase degradation of a fully-synthetic lung surfactant containing a novel diether phosphonolipid (DEPN-8) plus a 34 amino acid peptide (Mini-B) related to native surfactant protein (SP)-B. Activity studies used adsorption, pulsating bubble, and captive bubble methods to assess a range of surface behaviors, supplemented by molecular studies using Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD), and plasmon resonance. Calf lung surfactant extract (CLSE) was used as a positive control. Results DEPN-8+1.5% (by wt.) Mini-B was fully resistant to degradation by phospholipase A2 (PLA2) in vitro, while CLSE was severely degraded by this enzyme. Mini-B interacted with DEPN-8 at the molecular level based on FTIR spectroscopy, and had significant plasmon resonance binding affinity for DEPN-8. DEPN-8+1.5% Mini-B had greatly increased adsorption compared to DEPN-8 alone, but did not fully equal the very high adsorption of CLSE. In pulsating bubble studies at a low phospholipid concentration of 0.5 mg/ml, DEPN-8+1.5% Mini-B and CLSE both reached minimum surface tensions <1 mN/m after 10 min of cycling. DEPN-8 (2.5 mg/ml)+1.5% Mini-B and CLSE (2.5 mg/ml) also reached minimum surface tensions <1 mN/m at 10 min of pulsation in the presence of serum albumin (3 mg/ml) on the pulsating bubble. In captive bubble studies, DEPN-8+1.5% Mini-B and CLSE both generated minimum surface tensions <1 mN/m on 10 successive cycles of compression/expansion at quasi-static and dynamic rates. Conclusions These results show that DEPN-8 and 1.5% Mini-B form an interactive binary molecular mixture with very high surface activity and the ability to resist degradation by phospholipases in inflammatory lung injury. These characteristics are promising for the development of related fully-synthetic lipid/peptide exogenous surfactants for treating diseases of surfactant deficiency or dysfunction.

Surfactant with SP-B and SP-C analogues improves lung function in surfactant-deficient rats

The use of mammalian lung surfactant extracts has sharply reduced mortality and morbidity from respiratory distress syndrome in premature infants. Synthesis of surfactant protein B and C (SP-B and SP-C) analogues may lead the way to a synthetic surfactant preparation. Dimeric SP-B1–25 (dSP-B1–25) is based on the N-terminal domain of human SP-B and SP-Cfc is a modified human SP-C in which a single phenylalanine is substituted for a palmitoylated cysteine residue in the N-terminal segment (Phe-4 > Cys-4 variant). We tested the effects of synthetic surfactants with 1 or 2% dSP-B1–25 and 1% SP-Cfc on lung function in surfactant-deficient rats. Four experimental surfactant preparations were prepared by mixing 1% dSP-B1–25, 2% dSP-B1–25, 1% dSP-B1–25 +1% SP-Cfc, and 2% dSP-B1–25 +1% SP-Cfc with phospholipids (PL). PL and Survanta, a bovine lung extract, were controls. Groups of 8 rats were ventilated, lavaged until surfactant deficiency, and treated with 100 mg/kg surfactant. Arterial blood gas values and dynamic compliance were measured every 15 min and after 2 h of ventilation, the rats were killed and pressure-volume curves performed. Oxygenation improved quickly after instillation of surfactant with synthetic peptides and Survanta. Oxygenation and lung volumes were consistently higher in the 2% than in the 1% dSP-B1–25 groups. Addition of 1% SP-Cfc to the synthetic surfactants further improved oxygenation and lung volume, but to a lesser extent than increasing the dSP-B1–25 content from 1 to 2%. These data indicate that improvements in oxygenation and lung volume in lavaged rats are dependent on the concentration of dSP-B1–25 in the surfactant preparation and that the presence of SP-Cfc has a relative minor effect on these parameters.

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.

Comparison of three lipid formulations for synthetic surfactant with a surfactant protein B analog.

Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic dimeric SP-B1–25 (SP-B1–25), a peptide based on the N-terminal domain of human SP-B, efficiently mimics the functional properties of SP-B. The authors investigated the optimum lipid composition for SP-B1–25 by comparing the effects of natural lung lavage lipids (NLL), a synthetic equivalent of NLL (synthetic lavage lipids SLL), and a standard lipid mixture (TL) on the activities of SP-B1–25. Surfactant preparations were formulated by mixing 2 mol% SP-B1–25 in NNL, SLL, and TL. Calfactant, a calf lung lavage extract with SP-B and SP-C, was a positive control and lipids without peptide were negative controls. Minimum surface tension measured on a captive bubble surfactometer was similar for the three SP-B1–25 surfactant preparations and calfactant. The effects on lung function were compared in ventilated, lavaged, surfactant-deficient rats. Oxygenation and lung volumes were consistently higher in rats treated with calfactant and SP-B1–25 in NLL or SLL than in rats treated with SP-B1–25 in TL. Fourier transform infrared spectra observed abnormal secondary conformations for SP-B1–25 in TL as a possible cause for the reduced lung function. Lipid composition plays a crucial role in the in vitro and in vivo functions of SP-B1–25 in surfactant preparations.

Aerosol delivery of synthetic lung surfactant

Background. Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in premature infants with respiratory distress syndrome due to lack of lung surfactant. If this approach fails, the next step is often intubation, mechanical ventilation (MV) and intratracheal instillation of clinical lung surfactant. Objective. To investigate whether aerosol delivery of advanced synthetic lung surfactant, consisting of peptide mimics of surfactant proteins B and C (SP-B and SP-C) and synthetic lipids, during nCPAP improves lung function in surfactant-deficient rabbits. Methods. Experimental synthetic lung surfactants were produced by formulating 3% Super Mini-B peptide (SMB surfactant), a highly surface active SP-B mimic, and a combination of 1.5% SMB and 1.5% of the SP-C mimic SP-Css ion-lock 1 (BC surfactant), with a synthetic lipid mixture. After testing aerosol generation using a vibrating membrane nebulizer and aerosol conditioning (particle size, surfactant composition and surface activity), we investigated the effects of aerosol delivery of synthetic SMB and BC surfactant preparations on oxygenation and lung compliance in saline-lavaged, surfactant-deficient rabbits, supported with either nCPAP or MV. Results. Particle size distribution of the surfactant aerosols was within the 1–3 µm distribution range and surfactant activity was not affected by aerosolization. At a dose equivalent to clinical surfactant therapy in premature infants (100 mg/kg), aerosol delivery of both synthetic surfactant preparations led to a quick and clinically relevant improvement in oxygenation and lung compliance in the rabbits. Lung function recovered to a greater extent in rabbits supported with MV than with nCPAP. BC surfactant outperformed SMB surfactant in improving lung function and was associated with higher phospholipid values in bronchoalveolar lavage fluid; these findings were irrespective of the type of ventilatory support (nCPAP or MV) used. Conclusions. Aerosol delivery of synthetic lung surfactant with a combination of highly active second generation SP-B and SP-C mimics was effective as a therapeutic approach towards relieving surfactant deficiency in spontaneously breathing rabbits supported with nCPAP. To obtain similar results with nCPAP as with intratracheal instillation, higher dosage of synthetic surfactant and reduction of its retention by the delivery circuit will be needed to increase the lung dose.

Synthetic surfactant containing SP-B and SP-C mimics is superior to single-peptide formulations in rabbits with chemical acute lung injury

Background. Chemical spills are on the rise and inhalation of toxic chemicals may induce chemical acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). Although the pathophysiology of ALI/ARDS is well understood, the absence of specific antidotes has limited the effectiveness of therapeutic interventions. Objectives. Surfactant inactivation and formation of free radicals are important pathways in (chemical) ALI. We tested the potential of lipid mixtures with advanced surfactant protein B and C (SP-B and C) mimics to improve oxygenation and lung compliance in rabbits with lavage- and chemical-induced ALI/ARDS. Methods. Ventilated young adult rabbits underwent repeated saline lung lavages or underwent intratracheal instillation of hydrochloric acid to induce ALI/ARDS. After establishment of respiratory failure rabbits were treated with a single intratracheal dose of 100 mg/kg of synthetic surfactant composed of 3% Super Mini-B (S-MB), a SP-B mimic, and/or SP-C33 UCLA, a SP-C mimic, in a lipid mixture (DPPC:POPC:POPG 5:3:2 by weight), the clinical surfactant Infasurf®, a bovine lung lavage extract with SP-B and C, or synthetic lipids alone. End-points consisted of arterial oxygenation, dynamic lung compliance, and protein and lipid content in bronchoalveolar lavage fluid. Potential mechanism of surfactant action for S-MB and SP-C33 UCLA were investigated with captive bubble surfactometry (CBS) assays. Results. All three surfactant peptide/lipid mixtures and Infasurf equally lowered the minimum surface tension on CBS, and also improved oxygenation and lung compliance. In both animal models, the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA led to better arterial oxygenation and lung compliance than single peptide synthetic surfactants and Infasurf. Synthetic surfactants and Infasurf improved lung function further in lavage- than in chemical-induced respiratory failure, with the difference probably due to greater capillary-alveolar protein leakage and surfactant dysfunction after HCl instillation than following lung lavage. At the end of the duration of the experiments, synthetic surfactants provided more clinical stability in ALI/ARDS than Infasurf, and the protein content of bronchoalveolar lavage fluid was lowest for the two-peptide synthetic surfactant with S-MB and SP-C33 UCLA. Conclusion. Advanced synthetic surfactant with robust SP-B and SP-C mimics is better equipped to tackle surfactant inactivation in chemical ALI than synthetic surfactant with only a single surfactant peptide or animal-derived surfactant.

Surfactant protein C peptides with salt-bridges (“ion-locks”) promote high surfactant activities by mimicking the α-helix and membrane topography of the native protein

Background. Surfactant protein C (SP-C; 35 residues) in lungs has a cationic N-terminal domain with two cysteines covalently linked to palmitoyls and a C-terminal region enriched in Val, Leu and Ile. Native SP-C shows high surface activity, due to SP-C inserting in the bilayer with its cationic N-terminus binding to the polar headgroup and its hydrophobic C-terminus embedded as a tilted, transmembrane α-helix. The palmitoylcysteines in SP-C act as ‘helical adjuvants’ to maintain activity by overriding the β-sheet propensities of the native sequences. Objective. We studied SP-C peptides lacking palmitoyls, but containing glutamate and lysine at 4-residue intervals, to assess whether SP-C peptides with salt-bridges (“ion-locks”) promote surface activity by mimicking the α-helix and membrane topography of native SP-C. Methods. SP-C mimics were synthesized that reproduce native sequences, but without palmitoyls (i.e., SP-Css or SP-Cff, with serines or phenylalanines replacing the two cysteines). Ion-lock SP-C molecules were prepared by incorporating single or double Glu−–Lys+ into the parent SP-C’s. The secondary structures of SP-C mimics were studied with Fourier transform infrared (FTIR) spectroscopy and PASTA, an algorithm that predicts β-sheet propensities based on the energies of the various β-sheet pairings. The membrane topography of SP-C mimics was investigated with orientated and hydrogen/deuterium (H/D) exchange FTIR, and also Membrane Protein Explorer (MPEx) hydropathy analysis. In vitro surface activity was determined using adsorption surface pressure isotherms and captive bubble surfactometry, and in vivo surface activity from lung function measures in a rabbit model of surfactant deficiency. Results. PASTA calculations predicted that the SP-Css and SP-Cff peptides should each form parallel β-sheet aggregates, with FTIR spectroscopy confirming high parallel β-sheet with ‘amyloid-like’ properties. The enhanced β-sheet properties for SP-Css and SP-Cff are likely responsible for their low surfactant activities in the in vitro and in vivo assays. Although standard 12C-FTIR study showed that the α-helicity of these SP-C sequences in lipids was uniformly increased with Glu−–Lys+ insertions, elevated surfactant activity was only selectively observed. Additional results from oriented and H/D exchange FTIR experiments indicated that the high surfactant activities depend on the SP-C ion-locks recapitulating both the α-helicity and the membrane topography of native SP-C. SP-Css ion-lock 1, an SP-Css with a salt-bridge for a Glu−–Lys+ ion-pair predicted from MPEx hydropathy calculations, demonstrated enhanced surfactant activity and a transmembrane helix simulating those of native SP-C. Conclusion. Highly active SP-C mimics were developed that replace the palmitoyls of SP-C with intrapeptide salt-bridges and represent a new class of synthetic surfactants with therapeutic interest.

Synthetic lung surfactant reduces alveolar-capillary protein leakage in surfactant-deficient rabbits

ABSTRACT Purpose of the Study: Alveolar-capillary leakage of proteinaceous fluid impairs alveolar ventilation and surfactant function and decreases lung compliance in acute lung injury. We investigated the correlation between lung function and total protein levels in bronchoalveolar lavage fluid (BALF) of ventilated, lavaged surfactant-deficient rabbits treated with various clinical and synthetic lung surfactant preparations. Materials and Methods: 109 ventilated, young adult New Zealand White rabbits underwent lung lavage to induce surfactant-deficiency (PaO2 <100 torr in 100% O2), were treated with a clinical surfactant or a synthetic surfactant preparation with surfactant protein B (SP-B) and/or surfactant protein C (SP-C) analogs, and mechanically ventilated for 120 min. Total protein levels in postmortem BALF were correlated with arterial PO2 (PaO2) and dynamic lung compliance values at 120 min post-surfactant treatment. Results: Repeated lung lavages decreased mean PaO2 values from 540 to 58 torr and lung compliance from 0.64 to 0.33 mL/kg/cm H2O. Two hours after surfactant therapy and mechanical ventilation, mean PaO2 values had increased to 346 torr and lung compliance to 0.44 mL/kg/cm H2O. Eighty-six rabbits (79%) responded to surfactant therapy with an increase in PaO2 to values >200 torr. Fourteen non-responders received inactive surfactant preparations. BALF protein levels were inversely correlated with PaO2 and lung compliance (P < .001). Surfactant preparations containing both SP-B and SP-C proteins or peptide analogs outperformed single protein/peptide preparations. Conclusions: Clinical and synthetic surfactant therapy reduces alveolar-capillary protein leakage in surfactant-deficient rabbits. Surfactant preparations with both SP-B and SP-C (analogs) were more efficient than preparations with SP-B or SP-C alone.