Antimicrobial and biophysical properties of surfactant supplemented with an antimicrobial peptide for treatment of bacterial pneumonia.

Antibiotic-resistant bacterial infections represent an emerging health concern in clinical settings, and a lack of novel developments in the pharmaceutical pipeline is creating a "perfect storm" for multidrug-resistant bacterial infections. Antimicrobial peptides (AMPs) have been suggested as future therapeutics for these drug-resistant bacteria, since they have potent broad-spectrum activity, with little development of resistance. Due to the unique structure of the lung, bacterial pneumonia has the additional problem of delivering antimicrobials to the site of infection. One potential solution is coadministration of AMPs with exogenous surfactant, allowing for distribution of the peptides to distal airways and opening of collapsed lung regions. The objective of this study was to test various surfactant-AMP mixtures with regard to maintaining pulmonary surfactant biophysical properties and bactericidal functions. We compared the properties of four AMPs (CATH-1, CATH-2, CRAMP, and LL-37) suspended in bovine lipid-extract surfactant (BLES) by assessing surfactant-AMP mixture biophysical and antimicrobial functions. Antimicrobial activity was tested against methillicin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. All AMP/surfactant mixtures exhibited an increase of spreading compared to a BLES control. BLES+CATH-2 mixtures had no significantly different minimum surface tension versus the BLES control. Compared to the other cathelicidins, CATH-2 retained the most bactericidal activity in the presence of BLES. The BLES+CATH-2 mixture appears to be an optimal surfactant-AMP mixture based on in vitro assays. Future directions involve investigating the potential of this mixture in animal models of bacterial pneumonia.

Cholesterol-mediated surfactant dysfunction is mitigated by surfactant protein A.

The ability of pulmonary surfactant to reduce surface tension at the alveolar surface is impaired in various lung diseases. Recent animal studies indicate that elevated levels of cholesterol within surfactant may contribute to its inhibition. It was hypothesized that elevated cholesterol levels within surfactant inhibit human surfactant biophysical function and that these effects can be reversed by surfactant protein A (SP-A). The initial experiment examined the function of surfactant from mechanically ventilated trauma patients in the presence and absence of a cholesterol sequestering agent, methyl-ß-cyclodextrin. The results demonstrated improved surface activity when cholesterol was sequestered in vitro using a captive bubble surfactometer (CBS). These results were explored further by reconstitution of surfactant with various concentrations of cholesterol with and without SP-A, and testing of the functionality of these samples in vitro with the CBS and in vivo using surfactant depleted rats. Overall, the results consistently demonstrated that surfactant function was inhibited by levels of cholesterol of 10% (w/w phospholipid) but this inhibition was mitigated by the presence of SP-A. It is concluded that cholesterol-induced surfactant inhibition can actively contribute to physiological impairment of the lungs in mechanically ventilated patients and that SP-A levels may be important to maintain surfactant function in the presence of high cholesterol within surfactant.

A modified squeeze-out mechanism for generating high surface pressures with pulmonary surfactant.

The exact mechanism by which pulmonary surfactant films reach the very low surface tensions required to stabilize the alveoli at end expiration remains uncertain. We utilized the nanoscale sensitivity of atomic force microscopy (AFM) to examine phospholipid (PL) phase transition and multilayer formation for two Langmuir-Blodgett (LB) systems: a simple 3 PL surfactant-like mixture and the more complex bovine lipid extract surfactant (BLES). AFM height images demonstrated that both systems develop two types of liquid condensed (LC) domains (micro- and nano-sized) within a liquid expanded phase (LE). The 3 PL mixture failed to form significant multilayers at high surface pressure (π while BLES forms an extensive network of multilayer structures containing up to three bilayers. A close examination of the progression of multilayer formation reveals that multilayers start to form at the edge of the solid-like LC domains and also in the fluid-like LE phase. We used the elemental analysis capability of time-of-flight secondary ion mass spectrometry (ToF-SIMS) to show that multilayer structures are enriched in unsaturated PLs while the saturated PLs are concentrated in the remaining interfacial monolayer. This supports a modified squeeze-out model where film compression results in the hydrophobic surfactant protein-dependent formation of unsaturated PL-rich multilayers which remain functionally associated with a monolayer enriched in disaturated PL species. This allows the surface film to attain low surface tensions during compression and maintain values near equilibrium during expansion.

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 ToF-SIMS study of the lateral organization of lipids and proteins in pulmonary surfactant systems.

Pulmonary surfactant is a complex lipid-protein mixture whose main function is to reduce the surface tension at the air-liquid interface of alveoli to minimize the work of breathing. The exact mechanism by which surfactant monolayers and multilayers are formed and how they lower surface tension to very low values during lateral compression remains uncertain. We used time-of-flight secondary ion mass spectrometry to study the lateral organization of lipids and peptide in surfactant preparations ranging in complexity. We show that we can successfully determine the location of phospholipids, cholesterol and a peptide in surfactant Langmuir-Blodgett films and we can determine the effect of cholesterol and peptide addition. A thorough understanding of the lateral organization of PS interfacial films will aid in our understanding of the role of each component as well as different lipid-lipid and lipid-protein interactions. This may further our understanding of pulmonary surfactant function.

Chemical mapping of ceramide distribution in sphingomyelin-rich domains in monolayers.

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

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.

Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films. I. Micro- and nanostructures of functional pulmonary surfactant films and the effect of SP-A.

Monolayers of a functional pulmonary surfactant (PS) can reach very low surface tensions well below their equilibrium value. The mechanism by which PS monolayers reach such low surface tensions and maintain film stability remains unknown. As shown previously by fluorescence microscopy, phospholipid phase transition and separation seem to be important for the normal biophysical properties of PS. This work studied phospholipid phase transitions and separations in monolayers of bovine lipid extract surfactant using atomic force microscopy. Atomic force microscopy showed phospholipid phase separation on film compression and a monolayer-to-multilayer transition at surface pressure 40-50 mN/m. The tilted-condensed phase consisted of domains not only on the micrometer scale, as detected previously by fluorescence microscopy, but also on the nanometer scale, which is below the resolution limits of conventional optical methods. The nanodomains were embedded uniformly within the liquid-expanded phase. On compression, the microdomains broke up into nanodomains, thereby appearing to contribute to tilted-condensed and liquid-expanded phase remixing. Addition of surfactant protein A altered primarily the nanodomains and promoted the formation of multilayers. We conclude that the nanodomains play a predominant role in affecting the biophysical properties of PS monolayers and the monolayer-to-multilayer transition.

Studies of distribution, location and dynamic properties of EGFR on the cell surface measured by image correlation spectroscopy.

In this work, we have studied the distribution and dynamic properties of Epidermal Growth Factor (EGF) receptors in the plasma membrane of fixed and live cells as well as the extent of co-localization of this transmembrane protein with proteins specific for three-membrane microdomains: membrane rafts, caveolae and clathrin-coated pits. This was achieved using a family of image-processing tools called image correlation spectroscopy (ICS), image cross-correlation spectroscopy (ICCS) and dynamic image correlation spectroscopy (DICS). Our results indicate that EGFR is diffusely distributed on the cell surface at 37 degrees C and aggregates as the temperature is lowered to 4 degrees C. This aggregation takes place within 15 min and is reversible. Changes in temperature also affect the diffusion of EGFR by two orders of magnitude. The dynamic properties of EGFR are similar to the dynamic properties of a GPI-anchored protein known to be present in membrane rafts, which motivated us to explore the extent of co-localization of EGFR with this membrane raft protein using ICCS. Our results indicate that more than half of the EGFR population is present in membrane rafts and smaller percentages are present in caveolae and clathrin-coated pits.

Effect of cholesterol on the biophysical and physiological properties of a clinical pulmonary surfactant.

Pulmonary surfactant is a complex mixture of lipids and proteins that forms a surface-active film at the air-water interface of alveoli capable of reducing surface tension to near 0 mN/m. The role of cholesterol, the major neutral lipid component of pulmonary surfactant, remains uncertain. We studied the physiological effect of cholesterol by monitoring blood oxygenation levels of surfactant-deficient rats treated or not treated with bovine lipid extract surfactant (BLES) containing zero or physiological amounts of cholesterol. Our results indicate no significant difference between BLES and BLES containing cholesterol immediately after treatment; however, during ventilation, BLES-treated animals maintained higher PaO2 values compared to BLES+cholesterol-treated animals. We used a captive bubble tensiometer to show that physiological amounts of cholesterol do not have a detrimental effect on the surface activity of BLES at 37 degrees C. The effect of cholesterol on topography and lateral organization of BLES Langmuir-Blodgett films was also investigated using atomic force microscopy. Our data indicate that cholesterol induces the formation of domains within liquid-ordered domains (Lo). We used time-of-flight-secondary ion mass spectrometry and principal component analysis to show that cholesterol is concentrated in the Lo phase, where it induces structural changes.

Dynamics of GPI-anchored proteins on the surface of living cells.

Rather than being distributed homogeneously on the cell surface, proteins are probably aggregated in clusters or in specific domains. Some of these domains (lipid rafts) have lipid compositions, which differ from their surrounding membrane. They have been implicated in cell signaling, cell adhesion, and cholesterol homeostasis. Estimates of their size vary from 40 to 350 nm in diameter depending on the study and cell type used. Rafts are enriched in glycosphingolipids and cholesterol and appear to be in a more ordered lipid phase. Although there is some knowledge of their function in cell signaling, less is known about their assembly and dynamics in cells at various temperatures. We use image correlation spectroscopy and dynamic image correlation spectroscopy to study the clustering and diffusion of glycosylphosphatidylinositol (GPI)-anchored proteins within the plasma membrane of living cells at various temperatures. We find that GPI-anchored proteins occur both as monomers and in clusters at the cell surface. The propensities to cluster as well as the diffusion coefficient of these clusters are strongly temperature dependent. At 37 degrees C the GPI-anchored proteins are highly dynamic with a lower state of clustering than at lower temperatures.

Dynamics and interaction of caveolin-1 isoforms with BMP-receptors.

Caveolae are small invaginations of the cell membrane that are thought to play a role in important physiological functions such as cell surface signaling, endocytosis and intracellular cholesterol transport. Caveolin-1 is a key protein in these domains and contributes to the organization of cholesterol and saturated lipids within these vesicular invaginations of the plasma membrane. Caveolae are thought to be involved in the signaling of tyrosine kinase receptors and serine threonine receptors. In this article we focus on the involvement of caveolae in the signal transduction of bone morphogenetic proteins (BMPs). BMPs play important roles during embryonic development and especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. The initiation of the signal tranduction starts by the binding of a BMP to a corresponding set of BMP receptors. Using image cross-correlation spectroscopy, we show that the BMP receptors BRIa and BRII colocalize with caveolin-1 isoforms alpha and beta on the cell surface. BRIa colocalizes predominantly with the caveolin-1 alpha isoform. Coexpression of BRII leads to a redistribution of BRIa into domains enriched in caveolin-1 beta. After stimulation with BMP-2, BRIa moves back into the region with caveolin-1 alpha. BRII is expressed in regions enriched in caveolin-1 alpha and beta. Stimulation of cells with BMP-2 leads to a redistribution of BRII into domains enriched in caveolin-1 alpha. Immunoprecipitation studies using transfected COS-7 cells indicate that BRII binds to caveolin-1 alpha and beta. The binding of BRII to caveolin-1 was verified using A431 cells. Stimulation of starved A431 cells with BMP-2 lead to a release of caveolin-1 from the BMP receptors. We show further that the caveolin-1 beta isoform inhibits BMP signaling whereas the alpha isoform does not.

Caveolin-1 isoform reorganization studied by image correlation spectroscopy.

Caveolae are small, flask shaped invaginations in the cell membrane. They are thought to play a crucial role in cell signaling, endocytosis and intracellular cholesterol transport. Caveolin-1, 2 and 3 are key proteins, which are important for the formation of the invaginations on the cell surface. Caveolin-1 exists in two isoforms: caveolin-1 alpha (a) and caveolin-1 beta (beta). Little is known about the difference between these two isoforms, and less in known about their role in cell signaling. Bone morphogenetic proteins IBMPs) are a subfamily of the TGF beta superfamily and their response is mediated by serine/threonine kinase receptors. Epidermal growth factor (EGF) is known to signal through tyrosine kinase receptors of the ErbB family. Here we report on the aggregation and association of caveolin-1 isoforms with these receptors and the effect of BMP and EGF activation on caveolin-1 distribution in A431 cells. Our data, obtained by application of a family of image correlation spectroscopy tools, indicate that BMP and EGF stimulation lead to a rearrangement of the caveolin-1 isoforms on the cell surface. BMP as well as EGF stimulation leads to a rearrangement of the caveolin-1 P isoform into domains enriched in the caveolin-1 alpha isoform. We further show that about 20-30% of the caveolin-1 present at the surface of the cells co-localize with the EGF and BMP receptors. Using a reporter gene assay sensitive to the activation of the BMP pathway, we show that overexpression of caveolin-1beta inhibits signaling. Our data suggest that the two isoforms of caveolin-1 play different roles on the cell surface and that caveolae are dynamic structures.

Signal transduction of bone morphogenetic protein receptors.

Bone morphogenetic proteins (BMPs) play a crucial role during all stages of embryonic development. Although only two major signaling pathways have been characterized (the p38 and Smad pathways), the BMP signaling is complex and includes several negative feedback mechanisms. This article reviews the current state of BMP receptor signaling and provides a summary of the crosstalk of the BMP receptor pathway with other major signaling pathways.

Effect of the distribution and clustering of the type I A BMP receptor (ALK3) with the type II BMP receptor on the activation of signalling pathways.

Bone morphogenetic proteins (BMPs) play an important role during embryonic development, especially in chondrogenesis, osteogenesis, neurogenesis and hematopoiesis. There are over 19 BMPs known in mammalians, but only three BMP-type-I receptors and three BMP-type-II receptors are known so far to mediate these responses. Previous reports provide evidence to support that oligomerisation of BMP receptors influences the activation of the downstream BMP signalling pathways, the Smad or the p38 MAPK pathway. To further explore the importance of BMP receptor clustering in signalling, image correlation spectroscopy has been used to investigate the clustering and distribution of BMP receptors at the surface of the cell membrane. Here we demonstrate that the co-expression of the BMP-type-II receptor (BRII) influences the aggregation and the distribution of the BMP-type-Ia receptor (BRIa) in COS7 cells and in A431 cells. We also demonstrate that BMP-2 stimulation of the cells leads to a rearrangement of receptor complexes at the cell surface. Using A431 cells and limb bud-derived mesenchymal cells, we show that co-expression of the BRII and a constitutive active BRIa-ca is necessary for the activation of the Smad pathway. Importantly using a kinase-inactive BRII the rearrangement of BRIa is blocked. Together, these findings suggest that rearrangement of the receptors at the cell surface prior to forming preformed ligand independent complexes plays a critical role in activation of the Smad pathway. It also suggests further that the kinase activity of BRII is needed for signalling beyond the activation of BRIa at the GS domain.