Lung Surfactant Function Research Articles

Mechanistic Insights in the Interaction of Chemicals with Surfactant Membrane Models in vitro

Every year, cases of respiratory distress are recorded in humans following exposure to chemicals. The underlying mechanisms of toxicity remain unclear. We have previously shown that pulmonary surfactant is the target of impregnation spray products, leading to lung damage in humans. Lung surfactant is a lipid-protein mixture whose main function is to reduce the surface tension in the alveoli at the end of expiration, minimizing the work of breathing. In this study, three chemicals (trimethoxyoctylsilane, 3-oxo-2-pentyl-cyclopentaneacetic acid and diisoamyl ether), classified as acute inhalation toxicants and of relevance for worker and consumer exposure, were studied with respect to their potential to perturb lung surfactant function. Two models were used: DPPC, the most abundant surface active lipid in surfactant, as well as native surfactant purified from porcine bronchoalveolar lavage. The constrained drop surfactometer and the captive bubble surfactometer were used to assess the effects of the chemicals on the biophysical activity of the two surfactant models under different exposure conditions. These two methodologies mimic the breathing dynamics of the lungs. In addition, the effect of the chemicals on the lung surfactant structure was explored by (i) Langmuir-Blodgett balance experiments coupled with epifluorescence microscopy, and (ii) cryogenic transmission electron microscopy. Lastly, the thermotropic behavior of surfactant after exposure to the chemicals was investigated using differential scanning calorimetry. The data provide evidences on the mechanism of toxicity of chemicals potentially harmful for the lungs. The identification of key event(s) for the acute inhalation toxicity pathway is crucial for the design of relevant toxicity assays in vitro, which will contribute to having safer chemicals in the workplace and on the market.

Per- and polyfluoroalkyl substances (PFASs) modify lung surfactant function and pro-inflammatory responses in human bronchial epithelial cells

The toxicity of some per- and polyfluoroalkyl substances (PFASs), such as perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) has been studied thoroughly, showing that systemic PFASs targets the lungs. However, regulators lack data to assess the impact of other PFASs on the lungs and alternative methods to test substances for lung toxicity are needed. We combined two in vitro models to assess toxicity to the respiratory system; i) a lung surfactant (LS) function assay to assess the acute inhalation toxicity potential, and ii) a cell model with human bronchial epithelial cells to study pro-inflammatory potential and modulation of inflammatory responses. We tested salts of four PFASs: perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), PFOS, and PFOA as well as the fluorotelomer 8:2 FTOH. The results show that PFHxS, PFOA and PFOS can inhibit LS function. High PFOS concentrations induced a pro-inflammatory response, measured as increased IL-1α/β release. Moderate concentrations of PFOS suppressed release of the chemokines CXCL8 and CXCL10, whereas both PFOS and PFOA stimulated the release of the pro-inflammatory cytokine IL-1β in immune stimulated human bronchial epithelial cells. These findings support the concern that some PFASs may increase the risk of acute lung toxicity and of airway infections.

Lung morphology and surfactant function in cardiogenic pulmonary edema: a narrative review.

The conventional analysis of acute cardiogenic pulmonary edema involves the development of high pulmonary capillary pressures resulting in hydrostatic gradients for fluid flux out of capillaries into the interstitial space and alveolar spaces. However, some patients respond poorly to diuretic management. The PubMed database was searched to identify experimental studies on pulmonary edema in animals, experimental studies on surfactant function, including patients with pulmonary edema, and clinical studies reporting barrier dysfunction and/or injury in patients with acute pulmonary edema. Studies with animal models demonstrate that high capillary pressures can cause barrier disruption in alveolar capillary units which increases permeability and the transfer of fluid and protein into lung parenchyma. Fluid in alveolar spaces alters surfactant function which increases fluid flux out of capillaries into the lung parenchyma secondary to larger transcapillary hydrostatic gradients. Patients with acute cardiogenic pulmonary edema have increased levels of surfactant protein B in their plasma which reflect barrier disruption and increased levels of tumor necrosis factor alpha which reflect acute tissue injury. Increased surfactant protein B plasma levels are associated with abnormal gas exchange in patients with chronic heart failure. Patients with exercise-induced left ventricular dysfunction have increased levels of surfactant protein B after short periods of exercise. Pathology studies in patients with chronic heart failure have found increased connective tissue in alveolar capillary units and increased numbers of type II alveolar cells, and these changes represent an adaptive response in these patients. Clinicians need to consider the possibility of barrier dysfunction and disruption in patients with both acute and chronic pulmonary edema and understand that diuresis may have a limited effect on symptoms in some patients.

The influence of surfactant on the properties of albendazole-bile salts particles designed for lung delivery

Albendazole is a first line drug for the treatment of several parasitic diseases in humans. Some parasites target the lungs, however lung delivery of albendazole has so far not been reported. We have developed albendazole-based powders suitable for pulmonary delivery, and studied the impact of surfactant on the formulation properties. High-pressure homogenization followed by spray drying was used to produce inhalable particles of albendazole containing the bile salts sodium taurocholate and sodium glycocholate. The process resulted in porous microparticles, that exhibited good spray drying yield (>50%), low moisture contents (<1%) and aerodynamic D90 < 5 μm. The particles showed adequate aerosolization performance either for normal conditions (respiratory fraction > 71%) or conditions that simulate decreased respiratory capacity (respiratory fraction > 49%). The powders did not disturb lung surfactant function. The comparison between both formulations has revealed that the properties of the surfactants affect mainly the particle size of the suspensions and the porosity of the powders. The higher porosity of the albendazole-sodium taurocholate powder led to an enhanced aerodynamic performance of the formulation compared to albendazole-sodium glycocholate. The developed albendazole powders may pave a way for local lung treatment of parasitic diseases.

Bioinspired polymer nanoparticles omit biophysical interactions with natural lung surfactant

Herein, we report the attenuated impact of bioinspired nanoparticles on the essential function of lung surfactant. Colloidal particles made from poly(lactide) caused a significant loss of surfactant protein B (and C) from a natural lung surfactant accompanied by a decline in surface activity under static conditions and surface area cycling. No such perturbation of lung surfactant composition and function was observed for polymer nanoparticles coated with bioinspired poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC). More specifically, increasing the PMPC-coating layer thickness (≥3 nm) and density (dense conformation, distance of individual polymer chains of ≤3 nm) on the polymer nanoparticle surface diminished bioadverse events. PMPC-coated poly(lactide) nanoparticles provoked a less severe perturbation of the utilized lung surfactant when compared to colloidal counterparts coated with poly(ethylene glycol). Overall, a steric shielding of colloidal drug delivery vehicles with bioinspired PMPC can be considered as a valuable approach for the rationale development of biocompatible nanomedicines intended for lung delivery.

The influence of cannabis smoke and cannabis vapour on simulated lung surfactant function under physiologically relevant conditions

The use of cannabis for medicinal/recreational purposes is widespread throughout the world. Smoke inhalation is known to cause airway irritation due to noxious substances (ie, benzene) within the mix. Thus, advanced vaporisation platforms (eg, Davinci IQ) have been developed to circumvent negative health implications. Here, we consider the impact that cannabis smoke and cannabis vapour have on simulated lung surfactant performance within a model pulmonary space (ie, 37°C, elevated humidity and related fluid hydrodynamics). In total, 50 mg of herbal material was ignited or placed within a Davinci IQ vaporiser with subsequent activation. The aliquots were collected and then analysed using gas chromatography‐mass spectroscopy for composition and cannabinoid (eg, Δ9‐tetrahydrocannabinol [Δ9‐THC]) concentration. The average content within cannabis smoke was 2.84% (0.07%, SD) Δ9‐THC, with the same for cannabis vapour being 0.88% (0.14%, SD). Aerosolised samples were transferred to the lung biosimulator. When compared with the pristine Curosurf system, challenge with cannabis smoke and cannabis vapour reduced the surface pressure term by 26% and 7% and increased film compressibility by 60% and 15% at 80% trough area, respectively. The net effect would be enhanced film elasticity and an increased work of breathing, being more pronounced on cannabis smoke inhalation. The trends noted were ascribed to two factors operating synergistically, namely the amount of Δ9‐THC (plus others) within the aerosolised samples and the associated toxicity profile. Further research is required to establish mass‐balance effects (ie, titrated outputs) along with detailed chemical profiling of material generated from the unrelated cannabis activation pathways.

Bile salt enhancers for inhalation: Correlation between in vitro and in vivo lung effects

The lungs have potential as a means of systemic drug delivery of macromolecules. Systemic delivery requires crossing of the air-blood barrier, however with molecular size-dependent limitations in lung absorption of large molecules. Systemic availability after inhalation can be improved by absorption enhancers, such as bile salts. Enhancers may potentially interfere with the different constituents of the lungs, e.g. the lung surfactant lining the alveoli or the lung epithelium. We used two in vitro models to investigate the potential effects of bile salts on lung surfactant function (with the constrained drop surfactometer) and on the epithelium in the proximal airways (with the MucilAir™ cell system), respectively. In addition, we measured direct effects on respiration in mice inhaling bile salt aerosols. The bile salts inhibited lung surfactant function at different dose levels, however they did not affect the integrity of ciliated cells at the tested doses. Furthermore, the bile salt aerosols induced changes in the breathing pattern of mice indicative of pulmonary irritation. The bile salts were ranked according to potency in vitro for surfactant function disruption and in vivo for induction of pulmonary irritation. The ranking was the same, suggesting a correlation between the interference with lung surfactant and the respiratory response.

Compatibility of PEGylated Polymer Nanoparticles with the Biophysical Function of Lung Surfactant.

To minimize an unwanted interference of colloidal drug delivery vehicles with the biophysical functionality of lung surfactant, the surface of polymer nanoparticles was modified with poly(ethylene glycol) (PEGylation). Plain poly(lactide) nanoparticles provoked a statistically relevant decrease in the surface activity of the naturally derived lung surfactant, Alveofact. By contrast, the extent of lung surfactant inhibition induced by PEGylated polymer nanoparticles was significantly attenuated. Here, escalations of the PEG coating layer thickness (>3 nm, with a chain-to-chain distance of ≤4 nm) on the colloidal surface were capable of circumventing bioadverse effects. Accordingly, polymer nanoparticles equipped with PEG chains with a molecular weight above 2-5 kDa were compatible with the biophysical function of Alveofact. Overall, PEGylation of polymer nanoparticles presents a promising approach for the development of inhalation nanomedicines revealing negligible effects on the surface activity of the lining layer present in the deep lungs.

Electronic cigarette vapor alters the lateral structure but not tensiometric properties of calf lung surfactant

BackgroundDespite their growing popularity, the potential respiratory toxicity of electronic cigarettes (e-cigarettes) remains largely unknown. One potential aspect of e-cigarette toxicity is the effect of e-cigarette vapor on lung surfactant function. Lung surfactant is a mixture of lipids and proteins that lines the alveolar region. The surfactant layer reduces the surface tension of the alveolar fluid, thereby playing a crucial role in lung stability. Due to their small size, particulates in e-cigarette vapor can penetrate the deep lungs and come into contact with the lung surfactant. The current study sought to examine the potential adverse effects of e-cigarette vapor and conventional cigarette smoke on lung surfactant interfacial properties.MethodsInfasurf®, a clinically used and commercially available calf lung surfactant extract, was used as lung surfactant model. Infasurf® films were spread on top of an aqueous subphase in a Langmuir trough with smoke particulates from conventional cigarettes or vapor from different flavors of e-cigarettes dispersed in the subphase. Surfactant interfacial properties were measured in real-time upon surface compression while surfactant lateral structure after exposure to smoke or vapor was examined using atomic force microscopy (AFM).ResultsE-cigarette vapor regardless of the dose and flavoring of the e-liquid did not affect surfactant interfacial properties. In contrast, smoke from conventional cigarettes had a drastic, dose-dependent effect on Infasurf® interfacial properties reducing the maximum surface pressure from 65.1 ± 0.2 mN/m to 46.1 ± 1.3 mN/m at the highest dose. Cigarette smoke and e-cigarette vapor both altered surfactant microstructure resulting in an increase in the area of lipid multilayers. Studies with individual smoke components revealed that tar was the smoke component most disruptive to surfactant function.ConclusionsWhile both e-cigarette vapor and conventional cigarette smoke affect surfactant lateral structure, only cigarette smoke disrupts surfactant interfacial properties. The surfactant inhibitory compound in conventional cigarettes is tar, which is a product of burning and is thus absent in e-cigarette vapor.

Plasma lipidomics of tuberculosis patients: altered phosphatidylcholine remodeling.

Aim:Decreased circulating levels of lysophosphatidylcholines have been monitored in the serum of tuberculosis (TB) patients. However, the etiology of these findings has not been explored and other critical lung surfactant lipids have not been examined.Materials & methods:We undertook a lipidomics analysis of 30 controls and 30 TB patients, utilizing a high-resolution mass spectrometric analytical platform that assays over 1800 lipids.Findings:As previously reported, we found decrements in the plasma levels of lysophosphatidylcholines in TB patients. In addition, we report for the first time that there are increases in the plasma levels of phosphatidylcholines and phosphatidylglycerols in TB patients.Conclusion:These data suggest that TB results in altered glycerophosphocholine remodeling involving deacylation–reacylation reactions at sn-2 of the glycerol backbone. Such alterations in lipid remodeling have the potential to exert negative effects on the function of lung surfactant, on signal transduction mechanisms and membrane structural lipid architecture in TB patients.

Poloxamer-Decorated Polymer Nanoparticles for Lung Surfactant Compatibility.

Lung-delivered polymer nanoparticles provoked dysfunction of the essential lung surfactant system. A steric shielding of the nanoparticle surface with poloxamers could minimize the unwanted interference of polymer nanoparticles with the biophysical function of lung surfactant. The extent of poly(styrene) and poly(lactide) nanoparticle-induced lung surfactant inhibition could be related to the type and content of the applied poloxamer. Escalations of the adsorbed coating layer thickness (>3 nm) as well as concentration (brush- rather than mushroom-like conformation of poly(ethylene glycol), chain-to-chain distance of <5 nm) on the colloidal surface were capable of circumventing bioadverse effects. Accordingly, specific formulations (i.e., poloxamer 188, 338, and 407) avoided a perturbation of the microstructure and surface activity of Alveofact and a depletion of the content of surfactant-associated proteins. Poloxamer-modified polymer nanoparticles represent a promising nanomedicine platform intended for respiratory delivery revealing negligible effects on the biophysical functionality of the lining layer present in the deep lungs.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition.

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

Divide & Conquer: Surfactant Protein SP-C and Cholesterol Modulate Phase Segregation in Lung Surfactant

Lung surfactant (LS) is an essential system supporting the respiratory function. Cholesterol can be deleterious for LS function, a condition that is reversed by the presence of the lipopeptide SP-C. In this work, the structure of LS-mimicking membranes has been analyzed under the combined effect of SP-C and cholesterol by deuterium NMR and phosphorus NMR and by electron spin resonance. Our results show that SP-C induces phase segregation at 37°C, resulting in an ordered phase with spectral features resembling an interdigitated state enriched in dipalmitoylphosphatidylcholine, a liquid-crystalline bilayer phase, and an extremely mobile phase consistent with small vesicles or micelles. In the presence of cholesterol, POPC and POPG motion seem to be more hindered by SP-C than dipalmitoylphosphatidylcholine. The use of deuterated cholesterol did not show signs of specific interactions that could be attributed to SP-C or to the other hydrophobic surfactant protein SP-B. Palmitoylation of SP-C had an indirect effect on the extent of protein-lipid perturbations by stabilizing SP-C structure, and seemed to be important to maximize differences among the lipids participating in each phase. These results shed some light on how SP-C-induced lipid perturbations can alter membrane structure to sustain LS functionality at the air-liquid interface.

Impact of triblock copolymers on the biophysical function of naturally-derived lung surfactant

The current study aimed at investigating the general applicability of triblock copolymers consisting of poly(ethylene glycol) and poly(propylene glycol) (Pluronic®) as excipients for lung delivery. After thorough physicochemical characterization of the diverse polymers, their cytotoxicity was evaluated using alveolar epithelial cells. Next, a naturally-derived lung surfactant was challenged with the distinct triblock copolymers with respect to changes in microstructure, adsorption to the air/liquid interface and dynamic surface tension behavior under bubble pulsation.Biocompatibility assessment of triblock copolymers in A549 cells demonstrated some cytotoxicity, dependent on the hydrophobicity and dose of the substance applied (effective at ≥0.1mg/ml). Supplementing triblock copolymers onto Alveofact® had an obvious influence on the aggregation state and surface activity (>25 and >5mN/m during adsorption and bubble pulsation, respectively) of the lung surfactant. Interestingly, Pluronic® F127, a rather hydrophilic triblock copolymer, showed the most intense effect on the microstructure and biophysical performance of Alveofact®. This is likely due to the synergistic interplay of its low critical micelle concentration and rather high molecular weight, leading to the penetration of lung surfactant film/vesicles and accompanied by a partial replacement of relevant surfactant components from the air/liquid interface.Overall, suitable compositions and concentrations of triblock copolymers were identified with respect to compatibility with the physiological environment of the deep lungs.

Prediction of acute inhalation toxicity using in vitro lung surfactant inhibition

Private consumers and professionals may experience acute inhalation toxicity after inhaling aerosolized impregnation products. The distinction between toxic and non-toxic products is difficult to make for producers and product users alike, as there is no clearly described relationship between the chemical composition of the products and induction of toxicity. The currently accepted method for determination of acute inhalation toxicity is based on experiments on animals; it is time-consuming, expensive and causes stress for the animals. Impregnation products are present on the market in large numbers and amounts and exhibit great variety. Therefore, an alternative method to screen for acute inhalation toxicity is needed. The aim of our study was to determine if inhibition of lung surfactant by impregnation products in vitro could accurately predict toxicity in vivo in mice. We tested 21 impregnation products using the constant flow through set-up of the constrained drop surfactometer to determine if the products inhibited surfactant function or not. The same products were tested in a mouse inhalation bioassay to determine their toxicity in vivo. The sensitivity was 100%, i.e., the in vitro method predicted all the products that were toxic for mice to inhale. The specificity of the in vitro test was 63%, i.e., the in vitro method found three false positives in the 21 tested products. Six of the products had been involved in accidental human inhalation where they caused acute inhalation toxicity. All of these six products inhibited lung surfactant function in vitro and were toxic to mice.

A Proposed In Vitro Method to Assess Effects of Inhaled Particles on Lung Surfactant Function.

The lung surfactant (LS) lining is a thin liquid film covering the air-liquid interface of the respiratory tract. LS reduces surface tension, enabling lung surface expansion and contraction with minimal work during respiration. Disruption of surface tension is believed to play a key role in severe lung conditions. Inhalation of aerosols that interfere with the LS may induce a toxic response and, as a part of the safety assessment of chemicals and inhaled medicines, it may be relevant to study their impact on LS function. Here, we present a novel in vitro method, based on the constrained drop surfactometer, to study LS functionality after aerosol exposure. The applicability of the method was investigated using three inhaled asthma medicines, micronized lactose, a pharmaceutical excipient used in inhaled medication, and micronized albumin, a known inhibitor of surfactant function. The surfactometer was modified to allow particles mixed in air to flow through the chamber holding the surfactant drop. The deposited dose was measured with a custom-built quartz crystal microbalance. The alterations allowed the study of continuously increasing quantified doses of particles, allowing determination of the dose of particles that affects the LS function. The tested pharmaceuticals did not inhibit the function of a model LS even at extreme doses--neither did lactose. Micronized albumin, however, impaired surfactant function. The method can discriminate between safe inhaled aerosols--as exemplified by the approved inhaled medicines and the pharmaceutical excipient lactose--and albumin known to impair lung functionality by inhibiting LS function.

Computer simulations of lung surfactant

Lung surfactant lines the gas-exchange interface in the lungs and reduces the surface tension, which is necessary for breathing. Lung surfactant consists mainly of lipids with a small amount of proteins and forms a monolayer at the air–water interface connected to bilayer reservoirs. Lung surfactant function involves transfer of material between the monolayer and bilayers during the breathing cycle. Lipids and proteins are organized laterally in the monolayer; selected species are possibly preferentially transferred to bilayers. The complex 3D structure of lung surfactant and the exact roles of lipid organization and proteins remain important goals for research. We review recent simulation studies on the properties of lipid monolayers, monolayers with phase coexistence, monolayer–bilayer transformations, lipid–protein interactions, and effects of nanoparticles on lung surfactant. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Effect of clay nanoparticles on model lung surfactant: a potential marker of hazard from nanoaerosol inhalation.

This work investigates influence of different aluminosillicate nanoparticles (NPs) which are found in air in selected workplaces on the properties of the phospholipid (DPPC) monolayer at air–saline interface considered as ex vivo model of the lung surfactant (LS). The measurements were done under physiological-like conditions (deformable liquid interface at 37 °C) for NP concentrations matching the calculated lung doses after exposure in the working environment. Measured surface pressure–area (π–A) isotherms and compressibility curves demonstrated NP-induced changes in the structure and mechanical properties of the lipid monolayer. It was shown that hydrophilic nanomaterials (halloysite and bentonite) induced concentration-dependent impairment of DPPC’s ability of attaining high surface pressures on interfacial compression, suggesting a possibility of reduction of physiological function of natural LS. Hydrophobic montmorillonites affected DPPC monolayer in the opposite way; however, they significantly changed the mechanical properties of the air–liquid interface during compression. The results support the hypothesis of possible reduction or even degradation of the natural function of the lung surfactant induced by particle–phospholipid interactions after inhalation of nanoclays. Presented data do not only supplement the earlier results obtained with another LS model (animal-derived surfactant in oscillating bubble experiments) but also offer an explanation of physicochemical mechanisms responsible for detrimental effects which arise after deposition of inhaled nanomaterials on the surface of the respiratory system.

What We Talk About When We Talk About Evolution

Currently, the biologic sciences are a Tower of Babel, having become so highly specialized that one discipline cannot effectively communicate with another. A mechanism for evolution that integrates development and physiologic homeostasis phylogenetically has been identified—cell-cell interactions. By reducing this process to ligand-receptor interactions and their intermediate down-stream signaling partners, it is possible, for example, to envision the functional homologies between such seemingly disparate structures and functions as the lung alveolus and kidney glomerulus, the skin and brain, or the skin and lung. For example, by showing the continuum of the lung phenotype for gas exchange at the cell-molecular level, being selected for increased surface area by augmenting lung surfactant production and function in lowering surface tension, we have determined an unprecedented structural-functional continuum from proximate to ultimate causation in evolution. It is maintained that tracing the changes in structure and function that have occurred over both the short-term history of the organism (as ontogeny), and the long-term history of the organism (as phylogeny), and how the mechanisms shared in common can account for both biologic stability and novelty, will provide the key to understanding the mechanisms of evolution. We need to better understand evolution from its unicellular origins as the Big Bang of biology.

The Mechanism of Collapse of Heterogeneous Lipid Monolayers

Collapse of homogeneous lipid monolayers is known to proceed via wrinkling/buckling, followed by folding into bilayers in water. For heterogeneous monolayers with phase coexistence, the mechanism of collapse remains unclear. Here, we investigated collapse of lipid monolayers with coexisting liquid-liquid and liquid-solid domains using molecular dynamics simulations. The MARTINI coarse-grained model was employed to simulate monolayers of ∼80 nm in lateral dimension for 10–25 μs. The monolayer minimum surface tension decreased in the presence of solid domains, especially if they percolated. Liquid-ordered domains facilitated monolayer collapse due to the spontaneous curvature induced at a high cholesterol concentration. Upon collapse, bilayer folds formed in the liquid (disordered) phase; curved domains shifted the nucleation sites toward the phase boundary. The liquid (disordered) phase was preferentially transferred into bilayers, in agreement with the squeeze-out hypothesis. As a result, the composition and phase distribution were altered in the monolayer in equilibrium with bilayers compared to a flat monolayer at the same surface tension. The composition and phase behavior of the bilayers depended on the degree of monolayer compression. The monolayer-bilayer connection region was enriched in unsaturated lipids. Percolation of solid domains slowed down monolayer collapse by several orders of magnitude. These results are important for understanding the mechanism of two-to-three-dimensional transformations in heterogeneous thin films and the role of lateral organization in biological membranes. The study is directly relevant for the function of lung surfactant, and can explain the role of nanodomains in its surface activity and inhibition by an increased cholesterol concentration.