Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A

Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.     

Interfacial behavior and structural properties of a clinical lung surfactant from porcine source

Surfacen? is a clinical surfactant preparation of porcine origin, partly depleted of cholesterol, which is widely used in Cuba to treat pre-term babies at risk or already suffering neonatal respiratory distress. In the present study we have characterized the interfacial behavior of Surfacen in several in vitro functional models, including spreading and compression-expansion cycling isotherms in surface balances and in a captive bubble surfactometer, in comparison with the functional properties of whole native surfactant purified from porcine lungs and its reconstituted organic extract, the material from which Surfacen is derived. Surfacen exhibited similar properties to native porcine surfactant or its organic extract to efficiently form stable surface active films at the air-liquid interface, able to consistently reach surface tensions below 5mN/m upon repetitive compression-expansion cycling. Surfacen films, however, showed a substantially larger and stable compression-driven segregation of condensed lipid phases than exhibited by films formed by native surfactant or its organic extract. In spite of structural differences observed at microscopic level, Surfacen membranes showed a similar thermotropic behavior to membranes from native surfactant or its organic extract, characterized by calorimetry or fluorescence spectroscopy of samples doped with the Laurdan probe. On the other hand, analysis by atomic force microscopy of films formed by Surfacen or by the organic extract of native porcine surfactant revealed a similar network of interconnected condensed nanostructures, suggesting that the organization of the films at the submicroscopic level is the essential feature to support the proper stability and mechanical properties permitting the interfacial surfactant films to facilitate the work of breathing.     

Pulmonary Surfactant Protein SP-C Counteracts the Deleterious Effects of Cholesterol on the Activity of Surfactant Films under Physiologically Relevant Compression-Expansion Dynamics

The presence of cholesterol is critical in defining a dynamic lateral structure in pulmonary surfactant membranes. However, an excess of cholesterol has been associated with impaired surface activity of surfactant. It has also been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films. In this study, we analyzed the effect of SP-C on the thermodynamic properties of phospholipid membranes containing cholesterol, and the ability of lipid/protein complexes containing cholesterol to form and respread interfacial films capable of producing very low surface tensions upon repetitive compression-expansion cycling. SP-C modulates the effect of cholesterol to reduce the enthalpy associated with the gel-to-liquid-crystalline melting transition in dipalmitoylphosphatidylcholine (DPPC) bilayers, as analyzed by differential scanning calorimetry. The presence of SP-C affects more subtly the effects of cholesterol on the thermotropic properties of ternary membranes, mimicking more closely the lipid composition of native surfactant, where SP-C facilitates the miscibility of the sterol. Incorporation of 1% or 2% SP-C (protein/phospholipid by weight) promotes almost instantaneous adsorption of suspensions of DPPC/palmitoyloleoylphospatidylcholine (POPC)/palmitoyloleoyl-phosphatidylglycerol (POPG) (50:25:15, w/w/w) into the air-liquid interface of a captive bubble, in both the absence and presence of cholesterol. However, cholesterol impairs the ability of SP-C-containing films to achieve very low surface tensions in bubbles subjected to compression-expansion cycling. Cholesterol also substantially impairs the ability of DPPC/POPC/POPG films containing 1% surfactant protein SP-B to mimic the interfacial behavior of native surfactant films, which are characterized by very low minimum surface tensions with only limited area change during compression and practically no compression-expansion hysteresis. However, the simultaneous presence of 2% SP-C practically restores the compression-expansion dynamics of cholesterol- and SP-B-containing films to the efficient behavior shown in the absence of cholesterol. This suggests that cooperation between the two proteins is required for lipid-protein films containing cholesterol to achieve optimal performance under physiologically relevant compression-expansion dynamics.     

Meconium impairs pulmonary surfactant by a combined action of cholesterol and bile acids

Mechanisms for meconium-induced inactivation of pulmonary surfactant as part of the meconium aspiration syndrome in newborn infants, to our knowledge, are not clearly understood. Here we have studied the biophysical mechanisms of how meconium affects surface activity of pulmonary surfactant and whether the membrane-perturbing effects of meconium can be mimicked by exposure of surfactant to a mixture of bile acids and cholesterol. Surface activity of pulmonary surfactant complexes purified from animal lungs was analyzed in the absence and in the presence of meconium in standard surface balances and in a captive bubble surfactometer. We have also evaluated accumulation of surfactant at the air-liquid interface by what we believe to be a novel microtiter plate fluorescent assay, and the effect of meconium components on surfactant membrane fluidity using Laurdan fluorescence thermotropic profiles and differential scanning calorimetry thermograms. Rapid interfacial adsorption, low surface tension upon film compression, efficient film replenishment upon expansion, and thermotropic properties of surfactant complexes are all adversely affected by meconium, and, in a similar manner, they are affected by cholesterol/taurocholate mixtures but not by taurocholate alone. We conclude that inhibition of surfactant by meconium can be mimicked by a bile salt-promoted incorporation of excess cholesterol into surfactant complexes. These results highlight the potential pathogenic role of cholesterol-mobilizing agents as a crucial factor resulting in cholesterol induced alterations of structure and dynamics of surfactant membranes and films.     

Transient Exposure of Pulmonary Surfactant to Hyaluronan Promotes Structural and Compositional Transformations into a Highly Active State

Pulmonary surfactant is a lipid-protein complex that lowers surface tension at the respiratory air-liquid interface, stabilizing the lungs against physical forces tending to collapse alveoli. Dysfunction of surfactant is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome where naturally occurring surfactant-inhibitory agents such as serum, meconium, or cholesterol reach the lung. We analyzed the effect of hyaluronan (HA) on the structure and surface behavior of pulmonary surfactant to understand the mechanism for HA-promoted surfactant protection in the presence of inhibitory agents. In particular, we found that HA affects structural properties such as the aggregation state of surfactant membranes and the size, distribution, and order/packing of phase-segregated lipid domains. These effects do not require a direct interaction between surfactant complexes and HA and are accompanied by a compositional reorganization of large surfactant complexes that become enriched with saturated phospholipid species. HA-exposed surfactant reaches very high efficiency in terms of rapid and spontaneous adsorption of surfactant phospholipids at the air-liquid interface and shows significantly improved resistance to inactivation by serum or cholesterol. We propose that physical effects pertaining to the formation of a meshwork of interpenetrating HA polymer chains are responsible for the changes in surfactant structure and composition that enhance surfactant function and, thus, resistance to inactivation. The higher resistance of HA-exposed surfactant to inactivation persists even after removal of the polymer, suggesting that transient exposure of surfactant to polymers like HA could be a promising strategy for the production of more efficient therapeutic surfactant preparations.     

Palmitoylation of pulmonary surfactant protein SP-C is critical for its functional cooperation with SP-B to sustain compression/expansion dynamics in cholesterol-containing surfactant films

Recent data suggest that a functional cooperation between surfactant proteins SP-B and SP-C may be required to sustain a proper compression-expansion dynamics in the presence of physiological proportions of cholesterol. SP-C is a dually palmitoylated polypeptide of 4.2 kDa, but the role of acylation in SP-C activity is not completely understood. In this work we have compared the behavior of native palmitoylated SP-C and recombinant nonpalmitoylated versions of SP-C produced in bacteria to get a detailed insight into the importance of the palmitic chains to optimize interfacial performance of cholesterol-containing surfactant films. We found that palmitoylation of SP-C is not essential for the protein to promote rapid interfacial adsorption of phospholipids to equilibrium surface tensions (∼22 mN/m), in the presence or absence of cholesterol. However, palmitoylation of SP-C is critical for cholesterol-containing films to reach surface tensions ≤1 mN/m at the highest compression rates assessed in a captive bubble surfactometer, in the presence of SP-B. Interestingly, the ability of SP-C to facilitate reinsertion of phospholipids during expansion was not impaired to the same extent in the absence of palmitoylation, suggesting the existence of palmitoylation-dependent and -independent functions of the protein. We conclude that palmitoylation is key for the functional cooperation of SP-C with SP-B that enables cholesterol-containing surfactant films to reach very low tensions under compression, which could be particularly important in the design of clinical surfactants destined to replacement therapies in pathologies such as acute respiratory distress syndrome.     

Pulmonary surfactant proteins and polymer combinations reduce surfactant inhibition by serum

Acute respiratory distress syndrome (ARDS) is an inflammatory condition that can be associated with capillary leak of serum into alveoli causing inactivation of surfactant. Resistance to inactivation is affected by types and concentrations of surfactant proteins, lipids, and polymers. Our aim was to investigate the effects of different combinations of these three components. A simple lipid mixture (DPPC/POPG) or a more complex lipid mixture (DPPC/POPC/POPG/cholesterol) was used. Native surfactant proteins SP-B and SP-C obtained from pig lung lavage were added either singly or combined at two concentrations. Also, non-ionic polymers polyethylene glycol and dextran and the anionic polymer hyaluronan were added either singly or in pairs with hyaluronan included. Non-ionic polymers work by different mechanisms than anionic polymers, thus the purpose of placing them together in the same surfactant mixture was to evaluate if the combination would show enhanced beneficial effects. The resulting surfactant mixtures were studied in the presence or absence of serum. A modified bubble surfactometer was used to evaluate surface activities. Mixtures that included both SP-B and SP-C plus hyaluronan and either dextran or polyethylene glycol were found to be the most resistant to inhibition by serum. These mixtures, as well as some with either SP-B or SP-C with combined polymers were as or more resistant to inactivation than native surfactant. These results suggest that improved formulations of lung surfactants are possible and may be useful in reducing some types of surfactant inactivation in treating lung injuries.     

Intrinsic structural and functional determinants within the amino acid sequence of mature pulmonary surfactant protein SP-B

Pulmonary surfactant protein SP-B is absolutely required for proper function of surfactant in the alveoli, and is an important component of therapeutical surfactant preparations used to treat respiratory pathologies. To explore inherent structural and functional determinants within the amino acid sequence of mature SP-B, porcine SP-B has been subjected to extensive disulfide reduction under highly denaturing conditions and to cysteine carboxyamidomethylation, and the structure, lipid-protein interactions, and surface activity of this modified form have been characterized. Refolding of the reduced protein yielded a form (SP-Br) with secondary structure practically identical to that of the native disulfide-linked SP-B dimer. Reduced SP-Br exhibited higher structural flexibility than native SP-B, as indicated by a higher susceptibility of fluorescence emission to quenching by acrylamide and biphasic behavior during interaction of the protein with lipid bilayers and monolayers. SP-Br had, however, effects similar to those of native SP-B on the thermotropic properties of dipalmitoylphosphatidylcholine (DPPC) bilayers. SP-Br was more effective than native SP-B in promoting interfacial adsorption of phospholipid bilayers into interfacial films, presumably because of its higher structural flexibility, and retained the ability of native SP-B to stabilize DPPC interfacial films compressed to pressures near collapse against spontaneous relaxation. SP-Br also mimicked the behavior of native SP-B in lipid-protein films subjected to dynamic compression-expansion cycling in a captive bubble surfactometer, but only in the presence of phosphatidylglycerol (PG), the main anionic phospholipid in surfactant. The presence of PG appears to be required for SP-Br to acquire the appropriate tertiary folding to produce progressively more efficient lipid-protein films capable of reaching very high pressures upon limited compression with almost no hysteresis.     

Inactivation of pulmonary surfactant due to serum-inhibited adsorption and reversal by hydrophilic polymers: experimental

The rate of change of surface pressure, pi, in a Langmuir trough following the deposition of surfactant suspensions on subphases containing serum, with or without polymers, is used to model a likely cause of surfactant inactivation in vivo: inhibition of surfactant adsorption due to competitive adsorption of surface active serum proteins. Aqueous suspensions of native porcine surfactant, organic extracts of native surfactant, and the clinical surfactants Curosurf, Infasurf, and Survanta spread on buffered subphases increase the surface pressure, pi, to approximately 40 mN/m within 2 min. The variation with concentration, temperature, and mode of spreading confirmed Brewster angle microscopy observations that subphase to surface adsorption of surfactant is the dominant form of surfactant transport to the interface. However (with the exception of native porcine surfactant), similar rapid increases in pi did not occur when surfactants were applied to subphases containing serum. Components of serum are surface active and adsorb reversibly to the interface increasing pi up to a concentration-dependent saturation value, pi(max). When surfactants were applied to subphases containing serum, the increase in pi was significantly slowed or eliminated. Therefore, serum at the interface presents a barrier to surfactant adsorption. Addition of either hyaluronan (normally found in alveolar fluid) or polyethylene glycol to subphases containing serum reversed inhibition by restoring the rate of surfactant adsorption to that of the clean interface, thereby allowing surfactant to overcome the serum-induced barrier to adsorption.     

Methyl-β-cyclodextrin restores the structure and function of pulmonary surfactant films impaired by cholesterol

Pulmonary surfactant, a defined mixture of lipids and proteins, imparts very low surface tension to the lung-air interface by forming an incompressible film. In acute respiratory distress syndrome and other respiratory conditions, this function is impaired by a number of factors, among which is an increase of cholesterol in surfactant. The current study shows in vitro that cholesterol can be extracted from surfactant and function subsequently restored to dysfunctional surfactant films in a dose-dependent manner by methyl-β-cyclodextrin (MβCD). Bovine lipid extract surfactant was supplemented with cholesterol to serve as a model of dysfunctional surfactant. Likewise, when cholesterol in a complex with MβCD (“water-soluble cholesterol”) was added in aqueous solution, surfactant films were rendered dysfunctional. Atomic force microscopy showed recovery of function by MβCD is accompanied by the re-establishment of the native film structure of a lipid monolayer with scattered areas of lipid bilayer stacks, whereas dysfunctional films lacked bilayers. The current study expands upon a recent perspective of surfactant inactivation in disease and suggests a potential treatment.     

A comparative study of mechanisms of surfactant inhibition

Pulmonary surfactant spreads to the hydrated air-lung interface and reduces the surface tension to a very small value. This function fails in acute respiratory distress syndrome (ARDS) and the surface tension stays high. Dysfunction has been attributed to competition for the air-lung interface between plasma proteins and surfactant or, alternatively, to ARDS-specific alterations of the molecular profile of surfactant. Here, we compared the two mechanisms in vitro, to assess their potential role in causing respiratory distress. Albumin and fibrinogen exposure at or above blood level concentrations served as the models for testing competitive adsorption. An elevated level of cholesterol was chosen as a known adverse change in the molecular profile of surfactant in ARDS. Bovine lipid extract surfactant (BLES) was spread from a small bolus of a concentrated suspension (27 mg/ml) to the air-water interface in a captive bubble surfactometer (CBS) and the bubble volume was cyclically reduced and increased to assess surface activity of the spread material. Concentrations of inhibitors and the concentration and spreading method of pulmonary surfactant were chosen in an attempt to reproduce the exposure of surfactant to inhibitors in the lung. Under these conditions, neither serum albumin nor fibrinogen was persistently inhibitory and normal near-zero minimum surface tension values were obtained after a small number of cycles. In contrast, inhibition by an increased level of cholesterol persisted even after extensive cycling. These results suggest that in ARDS, competitive adsorption may not sufficiently explain high surface tension, and that disruption of the surfactant film needs to be given causal consideration.     

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic or anionic phospholipid monolayers. The peptide expands the pi-A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy. These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression-expansion cycling.     

Interaction of the N-terminal segment of pulmonary surfactant protein SP-C with interfacial phospholipid films

Pulmonary surfactant protein SP-C is a 35-residue polypeptide composed of a hydrophobic transmembrane alpha-helix and a polycationic, palmitoylated-cysteine containing N-terminal segment. This segment is likely the only structural motif the protein projects out of the bilayer in which SP-C is inserted and is therefore a candidate motif to participate in interactions with other bilayers or monolayers. In the present work, we have detected intrinsic ability of a peptide based on the sequence of the N-terminal segment of SP-C to interact and insert spontaneously into preformed zwitterionic or anionic phospholipid monolayers. The peptide expands the π-A compression isotherms of interfacial phospholipid/peptide films, and perturbs the lipid packing of phospholipid films during compression-driven liquid-expanded to liquid-condensed lateral transitions, as observed by epifluorescence microscopy. These results demonstrate that the sequence of the SP-C N-terminal region has intrinsic ability to interact with, insert into, and perturb the structure of zwitterionic and anionic phospholipid films, even in the absence of the palmitic chains attached to this segment in the native protein. This effect has been related with the ability of SP-C to facilitate reinsertion of surface active lipid molecules into the lung interface during respiratory compression-expansion cycling.     

Compositional,structural and functional properties of discrete coexisting complexes within bronchoalveolar pulmonary surfactant

Lung surfactant (LS) stabilizes the respiratory surface by forming a film at the alveolar air-liquid interface that reduces surface tension and minimizes the work of breathing. Typically, this surface-active agent has been isolated from animal lungs both for research and biomedical applications. However, these materials are constituted by complex membranous architectures including surface-active and inactive lipid/protein assemblies. In this work, we describe the composition, structure and surface activity of discrete membranous entities that are part of a LS preparation isolated from bronchoalveolar lavages of porcine lungs. Seven different fractions could be resolved from whole surfactant subjected to sucrose density gradient centrifugation. Detailed compositional characterization revealed differences in protein and cholesterol content but no distinct saturated:unsaturated phosphatidylcholine ratios. Moreover, no significant differences were detected regarding apparent hydration at the headgroup region of membranes, as reported by the probe Laurdan, and lipid chain mobility analysed by electron spin resonance (ESR) in spite of the variety of membranous assemblies observed by transmission electron microscopy. In addition, six of the seven separated LS subfractions formed similar, essentially disordered-like, interfacial films and performed efficient surface activity, under physiologically relevant conditions. Altogether, our work show that a LS isolated from porcine lungs is comprised by a heterogenous population of membranous assemblies lacking freshly secreted unused LS complexes sustaining highly dehydrated and ordered membranous assemblies as previously reported. We propose that surfactant subfractions may illustrate intermediates in sequential structural steps within the structural transformations occurring along the respiratory compression-expansion cycles.     

Visualizing the analogy between competitive adsorption and colloid stability to restore lung surfactant function

We investigated a model of acute respiratory distress syndrome in which the serum protein albumin adsorbs to an air-liquid interface and prevents the thermodynamically preferable adsorption of the clinical lung surfactant Survanta by inducing steric and electrostatic energy barriers analogous to those that prevent colloidal aggregation. Chitosan and polyethylene glycol (PEG), two polymers that traditionally have been used to aggregate colloids, both allow Survanta to quantitatively displace albumin from the interface, but through two distinct mechanisms. Direct visualization with confocal microscopy shows that the polycation chitosan coadsorbs to interfacial layers of both Survanta and albumin, and also colocalizes with the anionic domains of Survanta at the air-liquid interface, consistent with it eliminating the electrostatic repulsion by neutralizing the surface charges on albumin and Survanta. In contrast, the PEG distribution does not change during the displacement of albumin by Survanta, consistent with PEG inducing a depletion attraction sufficient to overcome the repulsive energy barrier toward adsorption.     

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.     

Overcoming rapid inactivation of lung surfactant: Analogies between competitive adsorption and colloid stability

Lung surfactant (LS) is a mixture of lipids and proteins that line the alveolar air-liquid interface, lowering the interfacial tension to levels that make breathing possible. In acute respiratory distress syndrome (ARDS), inactivation of LS is believed to play an important role in the development and severity of the disease. This review examines the competitive adsorption of LS and surface-active contaminants, such as serum proteins, present in the alveolar fluids of ARDS patients, and how this competitive adsorption can cause normal amounts of otherwise normal LS to be ineffective in lowering the interfacial tension. LS and serum proteins compete for the air-water interface when both are present in solution either in the alveolar fluids or in a Langmuir trough. Equilibrium favors LS as it has the lower equilibrium surface pressure, but the smaller proteins are kinetically favored over multi-micron LS bilayer aggregates by faster diffusion. If albumin reaches the interface, it creates an energy barrier to subsequent LS adsorption that slows or prevents the adsorption of the necessary amounts of LS required to lower surface tension. This process can be understood in terms of classic colloid stability theory in which an energy barrier to diffusion stabilizes colloidal suspensions against aggregation. This analogy provides qualitative and quantitative predictions regarding the origin of surfactant inactivation. An important corollary is that any additive that promotes colloid coagulation, such as increased electrolyte concentration, multivalent ions, hydrophilic non-adsorbing polymers such as PEG, dextran, etc. added to LS, or polyelectrolytes such as chitosan, also promotes LS adsorption in the presence of serum proteins and helps reverse surfactant inactivation. The theory provides quantitative tools to determine the optimal concentration of these additives and suggests that multiple additives may have a synergistic effect. A variety of physical and chemical techniques including isotherms, fluorescence microscopy, electron microscopy and X-ray diffraction show that LS adsorption is enhanced by this mechanism without substantially altering the structure or properties of the LS monolayer.     

An elevated level of cholesterol impairs self-assembly of pulmonary surfactant into a functional film

In adult respiratory distress syndrome, the primary function of pulmonary surfactant to strongly reduce the surface tension of the air-alveolar interface is impaired, resulting in diminished lung compliance, a decreased lung volume, and severe hypoxemia. Dysfunction coincides with an increased level of cholesterol in surfactant which on its own or together with other factors causes surfactant failure. In the current study, we investigated by atomic force microscopy and Kelvin-probe force microscopy how the increased level of cholesterol disrupts the assembly of an efficient film. Functional surfactant films underwent a monolayer-bilayer conversion upon contraction and resulted in a film with lipid bilayer stacks, scattered over a lipid monolayer. Large stacks were at positive electrical potential, small stacks at negative potential with respect to the surrounding monolayer areas. Dysfunctional films formed only few stacks. The surface potential of the occasional stacks was also not different from the surrounding monolayer. Based on film topology and potential distribution, we propose a mechanism for formation of stacked bilayer patches whereby the helical surfactant-associated protein SP-C becomes inserted into the bilayers with defined polarity. We discuss the functional role of the stacks as mechanically reinforcing elements and how an elevated level of cholesterol inhibits the formation of the stacks. This offers a simple biophysical explanation for surfactant inhibition in adult respiratory distress syndrome and possible targets for treatment.     

Pneumocytes Assemble Lung Surfactant as Highly Packed/Dehydrated States with Optimal Surface Activity

Pulmonary surfactant (PS) is an essential complex of lipids and specific proteins synthesized in alveolar type II pneumocytes, where it is assembled and stored intracellularly as multilayered organelles known as lamellar bodies (LBs). Once secreted upon physiological stimulation, LBs maintain a densely packed structure in the form of lamellar body-like particles (LBPs), which are efficiently transferred into the alveolar air-water interface, lowering surface tension to avoid lung collapse at end-expiration. In this work, the structural organization of membranes in LBs and LBPs freshly secreted by primary cultures of rat ATII cells has been compared with that of native lung surfactant membranes isolated from porcine bronchoalveolar lavage. PS assembles in LBs as crystalline-like highly ordered structures, with a highly packed and dehydrated state, which is maintained at supraphysiological temperatures. This relatively ordered/packed state is retained in secreted LBPs. The micro- and nanostructural examination of LBPs suggests the existence of high levels of structural complexity in comparison with the material purified from lavages, which may contain partially inactivated or spent structures. Additionally, freshly secreted surfactant LBPs exhibit superior activity when generating interfacial films and a higher intrinsic resistance to inactivating agents, such as serum proteins or meconium. We propose that LBs are assembled as an energy-activated structure competent to form very efficient interfacial films, and that the organization of lipids and proteins and the properties displayed by the films formed by LBPs are likely similar to those established at the alveolar interface and represent the actual functional structure of surfactant as it sustains respiration.     

Bacterial lipopolysaccharide promotes destabilization of lung surfactant-like films

The airspaces are lined with a dipalmitoylphosphatidylcholine (DPPC)-rich film called pulmonary surfactant, which is named for its ability to maintain normal respiratory mechanics by reducing surface tension at the air-liquid interface. Inhaled airborne particles containing bacterial lipopolysaccharide (LPS) may incorporate into the surfactant monolayer. In this study, we evaluated the effect of smooth LPS (S-LPS), containing the entire core oligosaccharide region and the O-antigen, on the biophysical properties of lung surfactant-like films composed of either DPPC or DPPC/palmitoyloleoylphosphatidylglycerol (POPG)/palmitic acid (PA) (28:9:5.6, w/w/w). Our results show that low amounts of S-LPS fluidized DPPC monolayers, as demonstrated by fluorescence microscopy and changes in the compressibility modulus. This promoted early collapse and prevented the attainment of high surface pressures. These destabilizing effects could not be relieved by repeated compression-expansion cycles. Similar effects were observed with surfactant-like films composed of DPPC/POPG/PA. On the other hand, the interaction of SP-A, a surfactant membrane-associated alveolar protein that also binds to LPS, with surfactant-like films containing S-LPS increased monolayer destabilization due to the extraction of lipid molecules from the monolayer, leading to the dissolution of monolayer material in the aqueous subphase. This suggests that SP-A may act as an LPS scavenger.