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.     

Protein denaturation by combined effect of shear and air-liquid interface

The effect of shear alone on the aggregation of recombinant human growth hormone (rhGH) and recombinant human deoxyribonuclease (rhDNase) has been found to be insignificant. This study focused on the synergetic effect of shear and gas-liquid interface on these two model proteins. Two shearing systems, the concentric-cylinder shear device (CCSD) and the rotor/stator homogenizer, were used to generate high shear (> 10(6)) in aqueous solutions in the presence of air. High shear in the presence of an air-liquid interface had no major effect on rhDNase but caused rhGH to form noncovalent aggregates. rhGH aggregation was induced by the air-liquid interface and was found to increase with increasing protein concentration and the air-liquid interfacial area. The aggregation was irreversible and exhibited a first-order kinetics with respect to the protein concentration and air-liquid interfacial area. Shear and shear rate enhanced the interaction because of its continuous generation of new air-liquid interfaces. In the presence of a surfactant, aggregation could be delayed or prevented depending upon the type and the concentration of the surfactant. The effect of air-liquid interface on proteins at low shear was examined using a nitrogen bubbling method. We found that foaming is very detrimental to rhGH even though the shear involved is low. The use of anti-foaming materials could prevent rhGH aggregation during bubbling. The superior stability exhibited by rhDNase may be linked to the higher surface tension and lower foaming tendency of its aqueous solution. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 503-512, 1997.     

Dynamic behavior of lung surfactant

We have previously developed an adsorption-limited model to describe the exchange of lung surfactant and its fractions to and from an air-liquid interface in oscillatory surfactometers. Here we extend this model to allow for diffusion in the liquid phase. Use of the model in conjunction with experimental data in the literature shows that diffusion-limited transport i.s important for characterizing the transient period from the start of oscillations to the achievement of steady-state conditions. Matching previous data shows that upon high levels of film compression, large changes occur in adsorption rate, desorption rate, and diffusion constant, consistent with what one might expect if the subsurface region was greatly enriched in DPPC. Collapse of the surfactant film that occurs during compression leads to a .significant elevation of surfactant concentration immediately heneath the interface, consistent with the subsurface depot of surfactant that has heen postulated by other investigators. Modeling studies also uncovered a phenomenon of surfactant behavior in which the interfacial tension remains constant at its minimum equilibrium value while the film is compressed, hut without collapse of the film. The phenomenon was due to desorption of surfactant from the interface and termed "pseudo-film collapse.' The new model also gave improved agreement with steady-state oscillatory cycling in a pulsating bubble surfactometer.     

Surface deactivation of cellulase and its prevention

When cellulase [1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] was exposed to air-liquid interface and subjected to shear, a significant deactivation was observed. The cellulase deactivation due to the interfacial effect combined with the shear effect was found to be far more severe and extensive than that due to the shear effect alone. Both increased cellulase concentration, and addition of surfactant (Zonyl or Triton) reduced the degree of deactivation. By using sufficient surfactant the cellulase deactivation can be prevented, and the cellulase can be stabilized and its use prolonged. The ratio of surface excess to the bulk protein is significantly reduced when the enzyme concentration is increased. The stabilizing effect of surfactant was attributed to the reduction in surface excess of cellulase.     

A biophysical mechanism by which plasma proteins inhibit lung surfactant activity

These in vitro experiments study a potential mechanism by which plasma proteins, found in the alveoli during pulmonary edema and hemorrhage, may act to inhibit the surface activity of pulmonary surfactant. The results indicate that the inhibition of the adsorption facility and surface tension lowering ability of a calf lung surfactant extract (CLSE) by albumin, hemoglobin, or fibrinogen may be completely abolished by centrifugation of the protein-surfactant mixture at 12,500 x g. Furthermore, albumin, hemoglobin and fibrinogen (1.25 mg/ml) were shown to inhibit the adsorption of high concentrations of CLSE (0.32 mg/ml), normally unaffected by the addition of exogenous proteins, when the CLSE was injected into the subphase under a preformed protein surface film. Similarly, injection of large amounts of these proteins (2.5 mg/ml) into the subphase beneath a preformed CLSE surface film was without effect, even though the CLSE concentration was only 0.06 mg/ml, a surfactant concentration which is normally inhibited by even small amounts of exogenous protein. Taken together, the data suggest that some proteins may inhibit surfactant function by preventing the surfactant phospholipids from adsorbing to the air-liquid interface, possibly by a competition between the proteins and CLSE phospholipids for space at the air-liquid interface rather than direct molecular interactions between proteins and surfactant.     

Investigation of air-liquid interfacial damage of animal cells in a falling film-flow device

A falling film-flow device was employed to expose animal cells to well-defined rapidly moving air-liquid interfaces. Cells (insect Sf-21) in the film flow suffered from severe mechanical damage with rapid cell death and lysis. The cell death rate was closely correlated with the momentum of the falling film but not the interfacial area, indicative of cell damage due to shear stress created by the falling film running into the bulk liquid, instead of direct interaction of the cells with the air-liquid interface.     

Optical measurement of surface tension in a miniaturized air-liquid interface and its application in lung physiology

We have previously shown that lamellar body-like particles, the form in which pulmonary surfactant is secreted, spontaneously disintegrate when they contact an air-liquid interface, eventually creating an interfacial film. Here, we combined these studies with a new technique enabling the simultaneous and non-invasive measurement of surface tension (gamma). This method is a refinement of the pendant-drop principle. A sapphire cone with a 300-microm aperture keeps the experimental fluid by virtue of surface coherence in a fixed and nearly planar position above the objective of an inverted microscope. The radius of curvature of the fluid meniscus is related to gamma and determines the pattern of light back-reflection upon epi-illumination. This method, which we name "inverted interface", has several novel aspects, in particular its microscopic dimensions. When using lamellar body-like particles freshly released by alveolar type II cells, we found that their conversion at the interface resulted in gamma-reduction close to 30 mN/m. After a fast initial decay, gamma-decrease proceeded slowly and in proportion to single particle conversions. These conversions ceased with time whereas gamma decreased further, probably due to reorganization of the already deposited material. The present investigation indicates that surface film formation by adsorption of large surfactant aggregates is an important mechanism by which gamma is reduced in the lung.     

Lamellar Bodies Form Solid Three-dimensional Films at the Respiratory Air-Liquid Interface

Pulmonary surfactant is essential for lung function. It is assembled, stored and secreted as particulate entities (lamellar body-like particles; LBPs). LBPs disintegrate when they contact an air-liquid interface, leading to an instantaneous spreading of material and a decline in surface tension. Here, we demonstrate that the film formed by the adsorbed material spontaneously segregate into distinct ordered and disordered lipid phase regions under unprecedented near-physiological conditions and, unlike natural surfactant purified from bronchoalveolar lavages, dynamically reorganized into highly viscous multilayer domains with complex three-dimensional topographies. Multilayer domains, in coexistence with liquid phases, showed a progressive stiffening and finally solidification, probably driven by a self-driven disassembly of LBPs from a sub-surface compartment. We conclude that surface film formation from LBPs is a highly dynamic and complex process, leading to a more elaborated scenario than that observed and predicted by models using reconstituted, lavaged, or fractionated preparations.     

Distinct steps in the adsorption of pulmonary surfactant to an air-liquid interface

To investigate the mechanisms by which vesicles of pulmonary surfactant adsorb to an air-liquid interface, we measured the effect of different phospholipids and of their concentration both in the subphase and at the interface on this process. Adsorbing vesicles contained the hydrophobic surfactant proteins mixed with the following four sets of surfactant phospholipids that varied the content of anionic headgroups and mixed acyl chains independently: the complete set of purified phospholipids (PPL) from calf surfactant; modified PPL (mPPL) from which the anionic phospholipids were removed; a mixture of dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylglycerol (DPPG) (9:1, mol:mol); and DPPC alone. The initial reduction in surface tension depended strongly on the anionic phospholipids and the subphase concentration. The acyl groups had no effect. Adsorption beyond the initial stage depended more on the mixed acyl groups, became increasingly independent of subphase concentration, and was determined instead by the interfacial concentration of the surface film. The different constituents produced the same effects in vesicles adsorbing to a clean interface or in a preexisting film to which vesicles of SP:DPPC adsorbed. Adsorption for vesicles of SP:PPL adsorbing to DPPC or of SP:DPPC to PPL above a certain threshold surface concentration followed exactly the same isotherm. Our results fit best with a two-step model for adsorption. The anionic phospholipids first promote the initial juxtaposition of vesicles to the interface. Compounds with mixed acyl constituents at the point of contact between vesicle and interface then facilitate fusion with the surface.     

Tracing surfactant transformation from cellular release to insertion into an air-liquid interface

Pulmonary surfactant is secreted by alveolar type II cells as lipid-rich, densely packed lamellar body-like particles (LBPs). The particulate nature of released LBPs might be the result of structural and/or thermodynamic forces. Thus mechanisms must exist that promote their transformation into functional units. To further define these mechanisms, we developed methods to follow LBPs from their release by cultured cells to insertion in an air-liquid interface. When released, LBPs underwent structural transformation, but did not disperse, and typically preserved a spherical appearance for days. Nevertheless, they were able to modify surface tension and exhibited high surface activity when measured with a capillary surfactometer. When LBPs inserted in an air-liquid interface were analyzed by fluorescence imaging microscopy, they showed remarkable structural transformations. These events were instantaneous but came to a halt when the interface was already occupied by previously transformed material or when surface tension was already low. These results suggest that the driving force for LBP transformation is determined by cohesive and tensile forces acting on these particles. They further suggest that transformation of LBPs is a self-regulated interfacial process that most likely does not require structural intermediates or enzymatic activation.     

Protein-lipid interactions and surface activity in the pulmonary surfactant system

Pulmonary surfactant is a lipid-protein complex, synthesized and secreted by the respiratory epithelium of lungs to the alveolar spaces, whose main function is to reduce the surface tension at the air-liquid interface to minimize the work of breathing. The activity of surfactant at the alveoli involves three main processes: (i) transfer of surface active molecules from the aqueous hypophase into the interface, (ii) surface tension reduction to values close to 0 mN/m during compression at expiration and (iii) re-extension of the surface active film upon expansion at inspiration. Phospholipids are the main surface active components of pulmonary surfactant, but the dynamic behaviour of phospholipids along the breathing cycle requires the necessary participation of some specific surfactant associated proteins. The present review summarizes the current knowledge on the structure, disposition and lipid-protein interactions of the hydrophobic surfactant proteins SP-B and SP-C, the two main actors participating in the surface properties of pulmonary surfactant. Some of the methodologies currently used to evaluate the surface activity of the proteins in lipid-protein surfactant preparations are also revised. Working models for the potential molecular mechanism of SP-B and SP-C are finally discussed. SP-B might act in surfactant as a sort of amphipathic tag, directing the lipid-protein complexes to insert and re-insert very efficiently into the air-liquid interface along successive breathing cycles. SP-C could be essential to maintain association of lipid-protein complexes with the interface at the highest compressed states, at the end of exhalation. The understanding of the mechanisms of action of these proteins is critical to approach the design and development of new clinical surfactant preparations for therapeutical applications.     

Structures of pulmonary surfactant films adsorbed to an air-liquid interface in vitro

Phospholipid films can be preserved in vitro when adsorbed to a solidifiable hypophase. Suspensions of natural surfactant, lipid extract surfactants, and artificial surfactants were added to a sodium alginate solution and filled into a captive bubble surfactometer (CBS). Surfactant film was formed by adsorption to the bubble of the CBS for functional tests. There were no discernible differences in adsorption, film compressibility or minimal surface tension on quasi-static or dynamic compression for films formed in the presence or absence of alginate in the subphase of the bubble. The hypophase-film complex was solidified by adding calcium ions to the suspension with the alginate. The preparations were stained with osmium tetroxide and uranyl acetate for transmission electron microscopy. The most noteworthy findings are: (1) Surfactants do adsorb to the surface of the bubble and form osmiophilic lining layers. Pure DPPC films could not be visualized. (2) A distinct structure of a particular surfactant film depends on the composition and the concentration of surfactant in the bulk phase, and on whether or not the films are compressed after their formation. The films appear heterogeneous, and frequent vesicular and multi-lamellar film segments are seen associated with the interfacial films. These features are seen already upon film formation by adsorption, but multi-lamellar segments are more frequent after film compression. (3) The rate of film formation, its compressibility, and the minimum surface tension achieved on film compression appear to be related to the film structure formed on adsorption, which in turn is related to the concentration of the surfactant suspension from which the film is formed. The osmiophilic surface associated surfactant material seen is likely important for the surface properties and the mechanical stability of the surfactant film at the air-fluid interface.     

Atomic force microscopy analysis of rat pulmonary surfactant films

Pulmonary surfactant facilitates breathing by forming a surface tension reducing film at the air-liquid interface of the alveoli. The objective was to characterize the structure of surfactant films using endogenous rat surfactant. Solid-support surfactant films, at different surface pressures, were obtained using a Langmuir balance and were analyzed using atomic force microscopy. The results showed a lipid film structure with three distinct phases: liquid expanded, liquid ordered and liquid condensed. The area covered by the liquid condensed domains increased as surface pressure increased. The presence of liquid ordered phase within these structures correlated with the cholesterol content. At a surface pressure of 50 mN/m, stacks of bilayers appeared. Several structural details of these films differ from previous observations made with goat and exogenous surfactants. Overall, the data indicate that surfactant films demonstrate phase separation at low surface pressures and multilayer formation at higher pressure, features likely important for normal surfactant function.     

A correlation between secondary structure and rheological properties of low-density lipoproteins at air/water interfaces

The secondary structure of apolipoprotein B-100 is studied within the bulk phase and at the air/water interface. In these “in viro” experiments, infrared reflection absorption spectroscopy (IRRAS) study was performed at the air/water interface while circular dichroism (CD) was conducted in the bulk phase. In the bulk phase, the conformational structure containing a significant amount of β–structure, whereas varying amount of α-helix, unordered structures, and β-sheet were observed at the air/water interface depending on the low-density lipoprotein (LDL) film interfacial pressure. The present IRRAS results demonstrate the importance of interfacial pressure-induced structural conformations on the apoB-100. A correlation between the secondary structure of the apoB-100 protein and the monomolecular film elasticity at the air/water interface was also established. The orientation of apoB-100 with respect to the LDL film-normal was found to depend on the interfacial pressure exhibited by the monomolecular film. These results may shed light on LDL’s pivotal role in the progression of atherosclerotic coronary artery disease as demonstrated previously by clinical trials.     

Role of lipid ordered/disordered phase coexistence in pulmonary surfactant function

The respiratory epithelium has evolved to produce a complicated network of extracellular membranes that are essential for breathing and, ultimately, survival. Surfactant membranes form a stable monolayer at the air-liquid interface with bilayer structures attached to it. By reducing the surface tension at the air-liquid interface, surfactant stabilizes the lung against collapse and facilitates inflation. The special composition of surfactant membranes results in the coexistence of two distinct micrometer-sized ordered/disordered phases maintained up to physiological temperatures. Phase coexistence might facilitate monolayer folding to form three-dimensional structures during exhalation and hence allow the film to attain minimal surface tension. These folded structures may act as a membrane reserve and attenuate the increase in membrane tension during inspiration. The present review summarizes what is known of ordered/disordered lipid phase coexistence in lung surfactant, paying attention to the possible role played by domain boundaries in the monolayer-to-multilayer transition, and the correlations of biophysical inactivation of pulmonary surfactant with alterations in phase coexistence.     

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.     

Comparison of simultaneous wet milling and enzymatic hydrolysis of cellulose in ball mill and attrition mill reactors

Simultaneous wet milling and enzymatic hydrolysis of newsprint in a ball mill reactor (BMR) and in an attrition mill reactor (AMR) were studied. The effect of milling on the enzyme deactivation was also investigated. Variables studied included milling media size, material, and the presence or absence of an air-liquid interface in the reactor. At a reaction temperature of 45 degrees C and a pH of 4.5 and in the presence of an air-liquid interface, the amount of reducing sugars produced by hydrolysis using a BMR was typically 5 to 15%less than the amount produced using an AMR. Without an air-liquid interface there was no significant difference. The effect of an air-;liquid interface on enzyme deactivation without any substrate was also studied. Enzyme activity after six hours of milling ranged from 1 to 85% ofthe starting activity. In both the AMR and the BMR, the extent of enzyme deactivation was 20 to 50% greater with an air-liquid interface present than with the air-liquid interface absent.     

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.     

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca²⁺ ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.     

Exposure to Polymers Reverses Inhibition of Pulmonary Surfactant by Serum,Meconium, or Cholesterol in the Captive Bubble Surfactometer

Dysfunction of pulmonary surfactant in the lungs is associated with respiratory pathologies such as acute respiratory distress syndrome or meconium aspiration syndrome. Serum, cholesterol, and meconium have been described as inhibitory agents of surfactant’s interfacial activity once these substances appear in alveolar spaces during lung injury and inflammation. The deleterious action of these agents has been only partly evaluated under physiologically relevant conditions. We have optimized a protocol to assess surfactant inhibition by serum, cholesterol, or meconium in the captive bubble surfactometer. Specific measures of surface activity before and after native surfactant was exposed to inhibitors included i), film formation, ii), readsorption of material from surface-associated reservoirs, and iii), interfacial film dynamics during compression-expansion cycling. Results show that serum creates a steric barrier that impedes surfactant reaching the interface. A mechanical perturbation of this barrier allows native surfactant to compete efficiently with serum to form a highly surface-active film. Exposure of native surfactant to cholesterol or meconium, on the other hand, modifies the compressibility of surfactant films though optimal compressibility properties recover on repetitive compression-expansion cycling. Addition of polymers like dextran or hyaluronic acid to surfactant fully reverses inhibition by serum. These polymers also prevent surfactant inhibition by cholesterol or meconium, suggesting that the protective action of polymers goes beyond the mere enhancement of interfacial adsorption as described by depletion force theories.