Pulmonary Surfactant Membranes Research Articles

Respiration: An Immiscibility Interfacial Phenomenon

Pulmonary surfactant (PS) is a surface active lipoproteic material produced by type II cells at the alveoli. This material forms a unique air-liquid interface lining the alveolar surface that reduces surface tension close to 0mN/m, maintaining lung volumes and alveolar homeostasis at the end of expiration. The particular lipid composition of PS suggests that mono- and bilayer-based structures should exhibit lateral phase segregation at physiological temperatures. This work on Native Pulmonary Surfactant Membranes (NPSM), directly isolated from broncho-alveolar lavages of wild-type mice show that a detailed lipid compositional study is crucial to understand the structure and biophysical function of these complex mixtures. Using different microscopy techniques, we have managed to analyze, in detail at the micro- and nano-scale, qualitative and quantitatively the whole native interfacial film responsible for breathing in mice. First, it was performed a study of the lipid phase segregation pattern on free-standing spherical and in planar solid-supported bilayers. Then, the phase behavior was investigated in planar solid-supported and in in-situ air-liquid monolayers. We found close correspondence in shape, size, height, area coverage and lipid phase of the domains between bilayers and monolayers. Contrary to what has been published until now. Particularly with monolayers, the quantification of the order among the different coexisting lipid phases indicates that the segregated rounded domains within a percolated larger area are more fluid. The phase segregation pattern remains until physiological relevant respiratory surface pressures, where we found non-homogeneous nanostructure serving as a platform for a highly corrugated-like collapsed structures, that grow size- and height-wise, arising from the more fluid phases. This last finding opens a new way to explore the pulmonary surfactant interfacial films, closer to materials science, which could help to understand such a basic and important topic; the respiration in mammals.

Fluid Pulmonary Surfactant Membranes of Torpid Animals Possess an Orderly Solid-Like Phase at Low Body Temperatures

Pulmonary surfactant (PS), a lipo-protein complex, regulates interfacial surface tension of the lung. The fluctuations in body temperatures of heterothermic mammals correlate with fluctuations in surfactant lipid composition as well as function. Previously we speculated that the higher levels of cholesterol during torpor will reduce phase transition temperature (Tm) enabling PS to remain fluid over a broader range of temperatures. However, these compositional changes do not explain how the surfactant can attain low surface tensions without suffering film collapse at the interface.

Phospholipid packing and hydration in pulmonary surfactant membranes and films as sensed by LAURDAN

The efficiency of pulmonary surfactant to stabilize the respiratory surface depends critically on the ability of surfactant to form highly packed films at the air–liquid interface. In the present study we have compared the packing and hydration properties of lipids in native pulmonary surfactant and in several surfactant models by analyzing the pressure and temperature dependence of the fluorescence emission of the LAURDAN (1-[6-(dimethylamino)-2-naphthyl]dodecan-1-one) probe incorporated into surfactant interfacial films or free-standing membranes. In interfacial films, compression-driven changes in the fluorescence of LAURDAN, evaluated from the generalized polarization function (GPF), correlated with changes in packing monitored by surface pressure. Compression isotherms and GPF profiles of films formed by native surfactant or its organic extract were compared at 25 or 37°C to those of films made of dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), DPPC/phosphatidylglycerol (PG) (7:3, w/w), or the mixture DPPC/POPC/palmitoyloleoylphosphatidylglycerol (POPG)/cholesterol (Chol) (50:25:15.10), which simulates the lipid composition of surfactant. In general terms, compression of surfactant films at 25°C leads to LAURDAN GPF values close to those obtained from pure DPPC monolayers, suggesting that compressed surfactant films reach a dehydrated state of the lipid surface, which is similar to that achieved in DPPC monolayers. However, at 37°C, the highest GPF values were achieved in films made of full surfactant organic extract or the mixture DPPC/POPC/POPG/Chol, suggesting a potentially important role of cholesterol to ensure maximal packing/dehydration under physiological constraints. Native surfactant films reached high pressures at 37°C while maintaining relatively low GPF, suggesting that the complex three-dimensional structures formed by whole surfactant might withstand the highest pressures without necessarily achieving full dehydration of the lipid environments sensed by LAURDAN. Finally, comparison of the thermotropic profiles of LAURDAN GPF in surfactant model bilayers and monolayers of analogous composition shows that the fluorophore probes an environment that is in average intrinsically more hydrated at the interface than inserted into free-standing bilayers, particularly at 37°C. This effect suggests that the dependence of membrane and surfactant events on the balance of polar/non-polar interactions could differ in bilayer and monolayer models, and might be affected differently by the access of water molecules to confined or free-standing lipid structures.

Lipid composition can predict mice native pulmonary surfactant membranes. 2. Fluid phase coexistence in bilayers and monolayers

Lipid composition can predict mice native pulmonary surfactant membranes. 2. Fluid phase coexistence in bilayers and monolayers

Native pulmonary surfactant membranes show similar phase segregation in bilayers and monolayers, both qualitatively and quantitatively, as predicted by lipid composition analysis

Native pulmonary surfactant membranes show similar phase segregation in bilayers and monolayers, both qualitatively and quantitatively, as predicted by lipid composition analysis

Pulmonary surfactant layers accelerate O2 diffusion through the air-water interface

During respiration, it is accepted that oxygen diffuses passively from the lung alveolar spaces through the respiratory epithelium until reaching the pulmonary capillaries, where blood is oxygenated. It is also widely assumed that pulmonary surfactant, a lipid-protein complex secreted into alveolar spaces, has a main surface active function, essential to stabilize the air-liquid interface, reducing in this way the work of breathing. The results of the present work show that capillary water layers containing enough density of pulmonary surfactant membranes transport oxygen much faster than a pure water phase or a water layer saturated with purely lipidic membranes. Membranes reconstituted from whole pulmonary surfactant organic extract, containing all the lipids plus the hydrophobic surfactant proteins, permit also very rapid oxygen diffusion, substantially faster than achieved by membranes prepared from the surfactant lipid fraction depleted of proteins. A model is proposed suggesting that protein-promoted membrane networks formed by pulmonary surfactant might have important properties to facilitate oxygenation through the thin water layer covering the respiratory surface.

Apoptosis induced by ozone and oxysterols in human alveolar epithelial cells

The mechanism of ozone-induced lung cell injury is poorly understood. One hypothesis is that ozone induces lipid peroxidation and that these peroxidated lipids produce oxidative stress and DNA damage. Oxysterols are lipid peroxides formed by the direct effects of ozone on pulmonary surfactant and cell membranes. We studied the effects of ozone and the oxysterol 5β,6β-epoxycholesterol (β-epoxide) and its metabolite cholestan-6-oxo-3,5-diol (6-oxo-3,5-diol) on human alveolar epithelial type I-like cells (ATI-like cells) and type II cells (ATII cells). Ozone and oxysterols induced apoptosis and cytotoxicity in ATI-like cells. They also generated reactive oxygen species and DNA damage. Ozone and β-epoxide were strong inducers of nuclear factor erythroid 2-related factor 2, heat shock protein 70, and Fos-related antigen 1 protein expression. Furthermore, we found higher sensitivity of ATI-like cells compared to ATII cells exposed to ozone or treated with β-epoxide or 6-oxo-3,5-diol. In general the response to the cholesterol epoxides was similar to the effect of ozone. Understanding the response of human ATI-like cells and ATII cells to oxysterols may be useful for further studies, because these compounds may represent useful biomarkers in other diseases.

SP-C Palmitoylation is Crucial for Stabilizing Cholesterol-Containing Surfactant Films during Continuous Compression/Expansion Cycling

Cholesterol is critical to maintain a dynamic lateral structure in pulmonary surfactant membranes, including a defined fluid-ordered/fluid-disordered phase equilibrium and proper lateral sorting of surfactant proteins and lipids. However, an excess of cholesterol has been linked to impaired surface activity both in surfactant models and in surfactant from injured lungs. Surfactant protein C (SP-C), the smallest and most hydrophobic of all surfactant proteins, has been shown to interact with cholesterol and dual palmitoylation of its N-terminal segment has been shown to drive association with ordered phases in model membranes. Furthermore, it has been proposed that native palmitoylated SP-C can act in concert with surfactant protein B (SP-B) to permit cholesterol-containing surfactant films to reach very low surface tensions upon compression. In the present work, we report that palmitoylation of SP-C is important for its ability to counteract deleterious effects of cholesterol on surfactant film stability under continuous expansion/compression cycling, as evaluated in a captive bubble surfactometer (CBS) setup. Presence of 5% cholesterol impairs significantly the stability under quasi static and dynamic compression of films composed of DPPC/POPC/POPG/SP-B (50:25:15:1, w/w/w/w), which are able to reach tensions below 3 mN/m with only 20% compression and almost no hysteresis in the absence of cholesterol. Incorporation in the films of 2% native palmitoylated SP-C could alleviate these deleterious effects. However, recombinant non-palmitoylated SP-C was not able to reproduce the stabilizing effect of native SP-C, confirming that palmitoylation of SP-C at its N-terminal end is crucial for its potential function of stabilizing surfactant films during the respiratory cycles in the lung.

Native Pulmonary Surfactant Membranes in Mice Show Coexistence of Two Different Phases in Bilayers and Monolayers: When the Lipid Composition can Predict the Structural Phase Segregations

Pulmonary surfactant is a surface active material composed both of lipids (aprox. 90% by weight) and proteins (aprox. 10%) produced by type II pneumocyte cells in the alveoli. This tension-active material forms a unique air-liquid interface at the alveolar cell surface that reduces surface tension close to 0 mN/m and maintains lung volumes and alveolar homeostasis at the end of the expiration. There are four pulmonary surfactant proteins (SP-A, -B, -C and -D). SP-A and -D have an important role in the immunological response against pathogens. The particular lipid composition of the lung surfactant suggests that surfactant mono- and bi-layer-based structures could exhibit lateral phase segregation at physiological temperatures. This work shows that in native pulmonary surfactant membranes a close lipid compositional study is crucial to understand the structure and biophysical function of these complex mixtures. Observing Giant Unilamellar Vesicles under conventional and two-photon excitation microscopy allow us to characterize and quantify the coexistence of two fluid-like phases in the wild-type (wt) native pulmonary surfactant membranes and a gel/fluid-like segregation pattern in the Knocked-out protein D (KOD) membranes. The atomic force microscopy studies of supported Langmuir-Blodgett bilayers and monolayers at different surface pressure show the same phase pattern before the collapse surface pressure of native pulmonary surfactant (∼40mN/m). Above this surface pressure different protruded structures can be observed arising from the more fluid phases. A closer look at the lipid composition reveals a higher content of saturated phospholipid species in the KOD native pulmonary surfactant membranes. This last finding explain the coexistence phenomena observed and allow us to conclude that the pulmonary surfactant segregation pattern could be predicted by an accurate lipid compositional study.

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.

Segregated Phases in Pulmonary Surfactant Membranes Do Not Show Coexistence of Lipid Populations with Differentiated Dynamic Properties

The composition of pulmonary surfactant membranes and films has evolved to support a complex lateral structure, including segregation of ordered/disordered phases maintained up to physiological temperatures. In this study, we have analyzed the temperature-dependent dynamic properties of native surfactant membranes and membranes reconstituted from two surfactant hydrophobic fractions (i.e., all the lipids plus the hydrophobic proteins SP-B and SP-C, or only the total lipid fraction). These preparations show micrometer-sized fluid ordered/disordered phase coexistence, associated with a broad endothermic transition ending close to 37°C. However, both types of membrane exhibit uniform lipid mobility when analyzed by electron paramagnetic resonance with different spin-labeled phospholipids. A similar feature is observed with pulse-field gradient NMR experiments on oriented membranes reconstituted from the two types of surfactant hydrophobic extract. These latter results suggest that lipid dynamics are similar in the coexisting fluid phases observed by fluorescence microscopy. Additionally, it is found that surfactant proteins significantly reduce the average intramolecular lipid mobility and translational diffusion of phospholipids in the membranes, and that removal of cholesterol has a profound impact on both the lateral structure and dynamics of surfactant lipid membranes. We believe that the particular lipid composition of surfactant imposes a highly dynamic framework on the membrane structure, as well as maintains a lateral organization that is poised at the edge of critical transitions occurring under physiological conditions.

Cholesterol modulates the exposure and orientation of pulmonary surfactant protein SP-C in model surfactant membranes

Cholesterol is the major neutral lipid in lung surfactant, accounting for up to 8–10% of surfactant mass, while surfactant protein SP-C (∼ 4.2 kDa) accounts for no more than 1–1.5% of total surfactant weight but plays critical roles in formation and stabilization of pulmonary surfactant films. It has been reported that surfactant protein SP-C interacts with cholesterol in lipid/protein interfacial films and this interaction could have a potential role on modulating surfactant function. In the present study, we have analyzed the effect of cholesterol on the structure, orientation and dynamic properties of SP-C embedded in physiologically relevant model membranes. The presence of cholesterol does not induce substantial changes in the secondary structure of SP-C, as analyzed by Attenuated Reflection Fourier Transformed Infrared spectroscopy (ATR-FTIR). However, the presence of cholesterol modifies the orientation of the transmembrane helix and the dynamic properties of the protein, as demonstrated by hydrogen/deuterium exchange kinetics. The effect of cholesterol on SP-C reconstituted in zwitterionic, entirely fluid, membranes made of POPC (palmitoyloleoylphospatidylcholine) or in anionic membranes with coexistence of ordered and disordered phases, such as those made of dipalmitoylphosphatidylcholine (DPPC):POPC:Palmitoyloleoylphosphatidylglycerol (POPG) (50:25:15) is dual. Cholesterol decreases the exposure of the protein to the aqueous environment and the tilt of its transmembrane helical segment up to a ratio Cholesterol:SP-C of 4.8 and 2.4 (mol/mol) in the two lipid systems tested, respectively, and it increases the exposure and tilt at higher cholesterol proportions. The results presented here suggest the existence of an interaction between SP-C and cholesterol-enriched phases, with consequences on the behavior of the protein, which could be of relevance for cholesterol-dependent structure–function relationships in pulmonary surfactant membranes and films.

Comparative Characterization of Lateral Organization and Packing Properties of Lipids in Pulmonary Surfactant Membranes and Interfacial Films

The main function of the lipid-protein complex pulmonary surfactant is to stabilize the respiratory surface by formation of a surface active interfacial film on top of the thin aqueous layer that covers the alveoli. From synthesis in the pulmonary epithelium, until formation of the functional film, surfactant complexes adopt different lamellar structures during its storage and secretion. The functional structure of surfactant makes Langmuir monolayers especially useful for the analysis of structure-function correlations in the pulmonary surfactant system, although they are also widely used as a model to obtain structural information about biological membranes. However, the exact correspondence between lateral organization and molecular packing in bilayers and monolayers is still a matter of controversy.The fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) is a lipophilic dye used to analyze the structure of membranes due to its spectral characteristics. Once inserted in membranes, the fluorescence emission of laurdan is very sensitive to the level of hydration of the phospholipid headgroups. Changes in packing or lateral organization of the membrane produce a shift of the emission maximum of the label from 440 nm in ordered membranes to 490 nm in disordered membrane phases. Taking advantage of the properties of this probe, films from pulmonary surfactant lipids containing laurdan were prepared and compressed to obtain surface pressure-area isotherms. Surface pressure and area occupied per molecule along compression were obtained in parallel with the generalized polarization function (GPF) of laurdan -as calculated from its interfacial fluorescence emission spectra- and compared with the fluorescence of laurdan in multilamellar suspensions of the same surfactant lipids at different temperatures.

Synthetic peptides representing the N-terminal segment of surfactant protein C modulate LPS-stimulated TNF-α production by macrophages

Surfactant protein C (SP-C) consists of a hydrophobic alpha-helix inserted in pulmonary surfactant membranes, and a more polar N-terminal palmitoylated segment exposed to the aqueous phase. Previously, we showed that SP-C inserted in lipid vesicles interacts with bacterial lipopolysaccharide (LPS) and reduces LPS-elicited responses. As the N-terminal segment of SP-C was the most likely region responsible for these effects, a set of synthetic analogs of this stretch (SPC((1-13)) ) were studied. Binding studies showed that SPC((1-13)) binds LPS to the same extent as porcine SP-C under lipid-free conditions. In the absence of serum, both, palmitoylated and non-palmitoylated analogs enhanced the binding of tritiated LPS to macrophages as well as the LPS-induced production of TNF-alpha by these cells. These effects were reversed in the presence of serum; the analogs reduced the production of TNF-alpha in LPS-stimulated macrophages, probably by interfering with the formation of LPS/CD14/LBP complexes as suggested by analysis of the fluorescence emitted by a FITC derivative of Re-LPS. Our data indicate that water-soluble analogs of the N-terminal segment of SP-C can reduce LPS effects in the presence of serum, and thus might help in the design of new derivatives to fight endotoxic shock and pro-inflammatory events.

Biophysical, Structural and Compositional characterization at the molecular level of Native Pulmonary Surfactant Membranes directly isolated from mice Wild-type and Knocked-out Protein D Bronco-alveolar Lavage Fluid

Pulmonary surfactant is a surface active material composed of lipids and proteins produced by type II pneumocytes cells in the alveoli. This tension-active material forms a unique interface separating gas and liquids at the alveolar cell surface, reduce surface tension close to 0mN/m and maintains lung volumes and alveolar homeostasis at the end of the expiration. Abnormalities of surfactant in the immature lung or in the acutely inflamed mature lung are related to several illnesses. There are four pulmonary surfactant proteins (SP-A, -B, -C and -D). SP-A and -D have a very important role in the immunological response against pathogens. The particular lipid composition of the lung surfactant suggests that native surfactant mono- and bi-layer-based structures could exhibit lateral segregation phenomena at physiological temperatures. The principles underlying the interfacial film and membrane-base organization are not well defined. Therefore, a deep study in the correlation among surfactant composition, structure and biophysical function is needed. The present work tries to get advantage of the combination of different biochemical and biophysical techniques applied to Native Pulmonary Surfactant Membranes directly isolated from wild-type and KO protein-D mice bronco-alveolar lavage fluid (BALF). Both mono- and bi-layers show the presence of different structural arrangements, which could indicate a phase lateral coexistence. A closer look at the lipid composition reveals several potential lipidic species which might be playing a role in the segregation phenomena in addition to SP-D. A detailed characterization and correlation with surfactant biophysical functional properties, as well as with particular molecular species is currently being performed. This experimental approach is extremely powerful to correlate the structural, biochemical, and biophysical properties of any compositionally complex material.

How does pulmonary surfactant reduce surface tension to very low values?

#### Background: Although initially proposed by von Neergaard in 1929 ([14][1]), direct evidence for surface-active material at the air-fluid interface of the lung was first reported by Richard Pattle and John Clements in the 1950s ([5][2], [10][3]). Pattle deduced that microbubbles formed from

Cholesterol Rules: DIRECT OBSERVATION OF THE COEXISTENCE OF TWO FLUID PHASES IN NATIVE PULMONARY SURFACTANT MEMBRANES AT PHYSIOLOGICAL TEMPERATURES

Pulmonary surfactant, the lipid-protein material that stabilizes the respiratory surface of the lungs, contains approximately equimolar amounts of saturated and unsaturated phospholipid species and significant proportions of cholesterol. Such lipid composition suggests that the membranes taking part in the surfactant structures could be organized heterogeneously in the form of inplane domains, originating from particular distributions of specific proteins and lipids. Here we report novel results concerning the lateral organization of bilayer membranes made of native pulmonary surfactant where the coexistence of two distinct micrometer sized fluid phases (fluid ordered and fluid disordered-like phases) is observed at physiological temperatures by using fluorescence microscopy and atomic force microscopy. Additional experiments using fluorescent-labeled proteins SP-B and SP-C show that at physiological temperatures these hydrophobic proteins are located exclusively in the fluid disordered-like phase. Most interestingly, the microscopic coexistence of fluid phases is maintained up to 37.5 degrees C, where most fluid ordered phases melt. This observation suggests that the particular composition of this material is naturally designed to be at the "edge" of a lateral structure transition under physiological conditions, likely providing particular structural and dynamic properties for its mechanical function. The observed lateral structure in native pulmonary surfactant membranes is dramatically affected by the extraction of cholesterol, an effect not observed upon extraction of the surfactant proteins. Furthermore, the spreading properties of the native surfactant material at the air-liquid interface were also greatly affected by cholesterol extraction, suggesting a connection between the observed lateral structure and a physiologically relevant function of the material. We suggest that the particular lipid composition of surfactant could be finely tuned to provide, under physiological conditions, a structural scaffold for surfactant proteins to act at appropriate local densities and lipid composition.

1689 REGULATION OF FATTY ACID SYNTHESIS IN DEVELOPING RAT

Fatty acids are integral components of both pulmonary surfactant and cell membranes. De novo fatty acid synthesis in adult tissues is regulated by the interplay of nutrition and hormones on the content of rate limiting enzymes, their state of phosphorylation and the concentration of allosteric effectors. To determine if de novo fatty acid synthesis in developing lung is regulated by similar factors, we measured the activity of the rate limiting enzyme acetyl-CoA carboxylase and its in vitro regulatory characteristics in fetal, newborn and adult rat lung. In fetal lung, acetyl-CoA carboxylase activity, measured in 100,000xg cell supernatant, increased from 1.01 nmoles/min/mg protein at 19 days gestation to 3.15 nmoles/min/mg protein by term (22 days). By one day after birth, activity fell to 30% of the peak fetal value. Activity remained low at 1, 2 and 3 weeks of age and in adult lung. In vitro activity of enzyme from fetal lung was greatly influenced by allosteric effectors: 2.0 mM citrate increased activity 5 fold while 25μM palmitoyl-CoA decreased activity by 75%. Pre-incubation in dephosphorylating conditions (10mM Mg++) resulted in a 50% increase in activity while phosphorylating conditions (2mM Mg++ - 4mM ATP) decreased activity 86%. These data demonstrate that acetyl-CoA carboxylase is very active in fetal lung, and the activity falls to low levels in the newborn period. In addition, in vitro regulation of the enzyme in fetal lung was qualitatively similar to that reported for adult liver, suggesting that fetal lung enzyme activity is modulated by similar mechanisms.

Dynamic compliance, limit cycles, and static equilibria of excised cat lung.

Dynamic compliance, limit cycles, and static equilibria of excised cat lung.