Inhibition of pulmonary surfactant adsorption by serum and the mechanisms of reversal by hydrophilic polymers: theory

A theory based on the Smolukowski analysis of colloid stability shows that the presence of charged, surface-active serum proteins at the alveolar air-liquid interface can severely reduce or eliminate the adsorption of lung surfactant from the subphase to the interface, consistent with the observations reported in the companion article (pages 1769-1779). Adding nonadsorbing, hydrophilic polymers to the subphase provides a depletion attraction between the surfactant aggregates and the interface, which can overcome the steric and electrostatic resistance to adsorption induced by serum. The depletion force increases with polymer concentration as well as with polymer molecular weight. Increasing the surfactant concentration has a much smaller effect than adding polymer, as is observed. Natural hydrophilic polymers, like the SP-A present in native surfactant, or hyaluronan, normally present in the alveolar fluids, can enhance adsorption in the presence of serum to eliminate inactivation.     

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.     

Surfactant protein A (SP-A): the alveolus and beyond.

Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air-liquid interface. The function of SP-A in the alveolus is to facilitate the surface tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function. It has also been shown that SP-A modulates host response to microbes and particulates at the level of the alveolus. More recently, several investigators have reported that pulmonary surfactant phospholipids and SP-A are present in nonalveolar pulmonary sites as well as in other organs of the body. We describe the structure and possible functions of alveolar SP-A as well as the sites of extra-alveolar SP-A expression and the possible functions of SP-A in these sites.     

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.     

Hyaluronan fragments: an information-rich system

Hyaluronan is a straight chain, glycosaminoglycan polymer of the extracellular matrix composed of repeating units of the disaccharide [-D-glucuronic acid-beta1,3-N-acetyl-D-glucosamine-beta1,4-]n. Hyaluronan is synthesized in mammals by at least three synthases with products of varying chain lengths. It has an extraordinary high rate of turnover with polymers being funneled through three catabolic pathways. At the cellular level, it is degraded progressively by a series of enzymatic reactions that generate polymers of decreasing sizes. Despite their exceedingly simple primary structure, hyaluronan fragments have extraordinarily wide-ranging and often opposing biological functions. There are large hyaluronan polymers that are space-filling, anti-angiogenic, immunosuppressive, and that impede differentiation, possibly by suppressing cell-cell interactions, or ligand access to cell surface receptors. Hyaluronan chains, which can reach 2 x 10(4) kDa in size, are involved in ovulation, embryogenesis, protection of epithelial layer integrity, wound repair, and regeneration. Smaller polysaccharide fragments are inflammatory, immuno-stimulatory and angiogenic. They can also compete with larger hyaluronan polymers for receptors. Low-molecular-size polymers appear to function as endogenous "danger signals", while even smaller fragments can ameliorate these effects. Tetrasaccharides, for example, are anti-apoptotic and inducers of heat shock proteins. Various fragments trigger different signal transduction pathways. Particular hyaluronan polysaccharides are also generated by malignant cells in order to co-opt normal cellular functions. How the small hyaluronan fragments are generated is unknown, nor is it established whether the enzymes of hyaluronan synthesis and degradation are involved in maintaining proper polymer sizes and concentration. The vast range of activities of hyaluronan polymers is reviewed here, in order to determine if patterns can be detected that would provide insight into their production and regulation.     

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

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

Effect of the phospholipid head group in antibiotic-phospholipid association at water-air interface

We studied the interactions of tetracycline antibiotics, TCs, with phospholipid monolayers with the two-fold aim of elucidating the mechanism of action of TCs and to provide a first step for the realization of bio-mimetic sensor for such drugs by means of the Langmuir-Blodgett technique. Preliminary surface tension studies demonstrated that surface activity of tetracycline is moderate and dependent on the pH of the subphase. We selected three phospholipids having hydrophobic chains of the same length but differing in the polar head structures, i.e. dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, and dipalmitoylphosphatidic acid. Surface pressure- and surface potential- area isotherms were employed to investigate the behavior of the phospholipid monolayers at the water-air interface when tetracycline was added to the aqueous subphase. Analysis of the results indicated that the electrostatic interaction is the driving force for migration of tetracycline towards the interface where localized adsorption to the head groups occurs. Nevertheless, such interactions appear to be insufficient to promote penetration of tetracycline through the hydrophobic layer.     

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

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

Are hyaluronan receptors involved in three-dimensional cell migration?

Hyaluronan (HA), an unbranched polysaccharide consisting of repeated glucuronic acid/N-acetylglucosamine disaccharide units, is ubiquitously present in the extracellular matrix of many tissues (for a more comprehensive review see: Fraser et al., 1997). Increased amounts of hyaluronan are produced by solid tumors and tumor-associated fibroblasts, and tumor-induced HA is correlated with poor prognosis. HA is well known to stimulate the migration of a large variety of cell types. Stimulation of cell migration by HA has been explained by different mechanisms. HA was shown to specifically bind to cell surface receptors, and inhibition of HA-receptor function was demonstrated to decrease cell migration and tumor growth. On the other hand, HA as a large hydrophilic molecule is also known to modulate the extracellular packing of collagen and fibrin, leading to increased fiber size and porosity of extracellular substrates. Hence a modified matrix architecture might similarly account for increased locomotion of cells. In this review, we attempted to summarize the available data on HA-induced cell migration, with particular emphasis on the role of HA receptors in three-dimensional cell migration. Although the HA receptor CD44 has been shown to mediate migration of cells over two-dimensional hyaluronan-coated surfaces in vitro, there is only little evidence that HA-binding to CD44 or other HA receptors has major impact on the locomotion of cells through three-dimensional matrices in vivo. We showed recently that the promigratory effect of HA in fibrin gels is largely due to HA-mediated modulation of fibrin polymerization. By increasing the porosity of fibrin gels, HA strongly accelerates cell migration. The porosity of matrices therefore appears as an important and probably underestimated determinant of cell migration and tumor spread.     

Highly efficient protein separations in high-performance capillary electrophoresis,using hydrophilic coatings on polysiloxane-bonded columns

Three methods for preparing hydrophilic coatings on polysiloxane bonded CElect H-type capillary electrophoresis columns have been shown. The polyalkylsiloxane-bonded phase is the first coating layer on the capillary surface, and nonionic surfactant, hydrophilic polymer, or polymer surfactant, adsorbed onto this first layer through hydrophobic interactions, forms the second coating layer. The resultant capillary surfaces are inert, hydrophilic, and suitable for highly efficient protein separations. The effectiveness and applicability of these capillary surface modification methods were tested for the separations of a variety of proteins over a wide range of buffer pH values under different capillary electrophoretic operation modes.     

Effects of gramicidin-A on the adsorption of phospholipids to the air-water interface

Prior studies suggest that the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of the lipids in pulmonary surfactant to an air-water interface by stabilizing a negatively curved rate-limiting structure that is intermediate between bilayer vesicles and the surface film. This model predicts that other peptides capable of stabilizing negative curvature should also promote lipid adsorption. Previous reports have shown that under appropriate conditions, gramicidin-A (GrA) induces dioleoyl phosphatidylcholine (DOPC), but not dimyristoyl phosphatidylcholine (DMPC), to form the negatively curved hexagonal-II (H_II) phase. The studies reported here determined if GrA would produce the same effects on adsorption of DMPC and DOPC that the hydrophobic surfactant proteins have on the surfactant lipids. Small angle X-ray scattering and ³¹P-nuclear magnetic resonance confirmed that at the particular conditions used to study adsorption, GrA induced DOPC to form the H_II phase, but DMPC remained lamellar. Measurements of surface tension showed that GrA in vesicles produced a general increase in the rate of adsorption for both phospholipids. When restricted to the interface, however, in preexisting films, GrA with DOPC, but not with DMPC, replicated the ability of the surfactant proteins to promote adsorption of vesicles containing only the lipids. The correlation between the structural and functional effects of GrA with the two phospholipids, and the similar effects on adsorption of GrA with DOPC and the hydrophobic surfactant proteins with the surfactant lipids fit with the model in which SP-B and SP-C facilitate adsorption by stabilizing a rate-limiting intermediate with negative curvature.     

Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury

The lung is composed of a series of branching conducting airways that terminate in grape-like clusters of delicate gas-exchanging airspaces called pulmonary alveoli. Maintenance of alveolar patency at end expiration requires pulmonary surfactant, a mixture of phospholipids and proteins that coats the epithelial surface and reduces surface tension. The surfactant lining is exposed to the highest ambient oxygen tension of any internal interface and encounters a variety of oxidizing toxicants including ozone and trace metals contained within the 10 kl of air that is respired daily. The pathophysiological consequences of surfactant oxidation in humans and experimental animals include airspace collapse, reduced lung compliance, and impaired gas exchange. We now report that the hydrophilic surfactant proteins A (SP-A) and D (SP-D) directly protect surfactant phospholipids and macrophages from oxidative damage. Both proteins block accumulation of thiobarbituric acid-reactive substances and conjugated dienes during copper-induced oxidation of surfactant lipids or low density lipoprotein particles by a mechanism that does not involve metal chelation or oxidative modification of the proteins. Low density lipoprotein oxidation is instantaneously arrested upon SP-A or SP-D addition, suggesting direct interference with free radical formation or propagation. The antioxidant activity of SP-A maps to the carboxyl-terminal domain of the protein, which, like SP-D, contains a C-type lectin carbohydrate recognition domain. These results indicate that SP-A and SP-D, which are ubiquitous among air breathing organisms, could contribute to the protection of the lung from oxidative stresses due to atmospheric or supplemental oxygen, air pollutants, and lung inflammation.     

Enhanced surfactant adsorption via polymer depletion forces: a simple model for reversing surfactant inhibition in acute respiratory distress syndrome

Lung surfactant adsorption to an air-water interface is strongly inhibited by an energy barrier imposed by the competitive adsorption of albumin and other surface-active serum proteins that are present in the lung during acute respiratory distress syndrome. This reduction in surfactant adsorption results in an increased surface tension in the lung and an increase in the work of breathing. The reduction in surfactant adsorption is quantitatively described using a variation of the classical Smolukowski analysis of colloid stability. Albumin adsorbed to the interface induces an energy barrier to surfactant diffusion of order 5 k(B)T, leading to a reduction in adsorption equivalent to reducing the surfactant concentration by a factor of 100. Adding hydrophilic, nonadsorbing polymers such as polyethylene glycol to the subphase provides a depletion attraction between the surfactant aggregates and the interface that eliminates the energy barrier. Surfactant adsorption increases exponentially with polymer concentration as predicted by the simple Asakura and Oosawa model of depletion attraction. Depletion forces can likely be used to overcome barriers to adsorption at a variety of liquid-vapor and solid-liquid interfaces.     

Hyaluronan fragments induce endothelial cell differentiation in a CD44- and CXCL1/GRO1-dependent manner

Hyaluronan is a glycosaminoglycan of the extracellular matrix. In tumors and during chronic inflammatory diseases, hyaluronan is degraded to smaller fragments, which are known to stimulate endothelial cell differentiation. In this study, we have compared the molecular mechanisms through which hyaluronan dodecasaccharides (HA12), and the known angiogenic factor, fibroblast growth factor 2 (FGF-2), induce capillary endothelial cell sprouting in a three-dimensional collagen gel. The gene expression profiles of unstimulated and HA12- or FGF-2-stimulated endothelial cells were compared using a microarray analysis approach. The data revealed that both FGF-2 and HA12 promoted endothelial cell morphogenesis in a process depending on the expression of ornithine decarboxylase (Odc) and ornithine decarboxylase antizyme inhibitor (Oazi) genes. Among the genes selectively up-regulated in response to HA12 was the chemokine CXCL1/GRO1 gene. The notion that the induction of CXCL1/GRO1 is of importance for HA12-induced endothelial cell sprouting was supported by the fact that morphogenesis was inhibited by antibodies specifically neutralizing the CXCL1/GRO1 protein product. HA12-stimulated endothelial cell differentiation was exerted via binding to CD44 since it was inhibited by antibodies blocking CD44 function. Our data show that hyaluronan fragments and FGF-2 affect endothelial cell morphogenesis by the induction of overlapping but also by distinct sets of genes.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

Expression and purification of RHC–EGFP fusion protein and its application in hyaluronic acid assay

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM–enhanced green fluorescence protein (RHC–EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC–EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.     

Effect of hydrophobic surfactant peptides SP-B and SP-C on binary phospholipid monolayers. I. Fluorescence and dark-field microscopy.

The influence of the hydrophobic proteins SP-B and SP-C, isolated from pulmonary surfactant, on the morphology of binary monomolecular lipid films containing phosphocholine and phosphoglycerol (DPPC and DPPG) at the air-water interface has been studied using epifluorescence and dark-field microscopy. In contrast to previously published studies, the monolayer experiments used the entire hydrophobic surfactant protein fraction (containing both the SP-B and SP-C peptides) at physiologically relevant concentrations (approximately 1 wt %). Even at such low levels, the SP-B/C peptides induce the formation of a new phase in the surface monolayer that is of lower intrinsic order than the liquid condensed (LC) phase that forms in the pure lipid mixture. This presumably leads to a higher structural flexibility of the surface monolayer at high lateral pressure. Variation of the subphase pH indicates that electrostatic interaction dominates the association of the SP-B/C peptides with the lipid monolayer. As evidenced from dark-field microscopy, monolayer material is excluded from the DPPC/DPPG surface film on compression and forms three-dimensional, surface-associated structures of micron dimensions. Such exclusion bodies formed only with SP-B/C peptides. This observation provides the first direct optical evidence for the squeeze-out of pulmonary surfactant material in situ at the air-water interface upon increasing monolayer surface pressures.     

The interplay of lung surfactant proteins and lipids assimilates the macrophage clearance of nanoparticles

The peripheral lungs are a potential entrance portal for nanoparticles into the human body due to their large surface area. The fact that nanoparticles can be deposited in the alveolar region of the lungs is of interest for pulmonary drug delivery strategies and is of equal importance for toxicological considerations. Therefore, a detailed understanding of nanoparticle interaction with the structures of this largest and most sensitive part of the lungs is important for both nanomedicine and nanotoxicology. Astonishingly, there is still little known about the bio-nano interactions that occur after nanoparticle deposition in the alveoli. In this study, we compared the effects of surfactant-associated protein A (SP-A) and D (SP-D) on the clearance of magnetite nanoparticles (mNP) with either more hydrophilic (starch) or hydrophobic (phosphatidylcholine) surface modification by an alveolar macrophage (AM) cell line (MH-S) using flow cytometry and confocal microscopy. Both proteins enhanced the AM uptake of mNP compared with pristine nanoparticles; for the hydrophilic ST-mNP, this effect was strongest with SP-D, whereas for the hydrophobic PL-mNP it was most pronounced with SP-A. Using gel electrophoretic and dynamic light scattering methods, we were able to demonstrate that the observed cellular effects were related to protein adsorption and to protein-mediated interference with the colloidal stability. Next, we investigated the influence of various surfactant lipids on nanoparticle uptake by AM because lipids are the major surfactant component. Synthetic surfactant lipid and isolated native surfactant preparations significantly modulated the effects exerted by SP-A and SP-D, respectively, resulting in comparable levels of macrophage interaction for both hydrophilic and hydrophobic nanoparticles. Our findings suggest that because of the interplay of both surfactant lipids and proteins, the AM clearance of nanoparticles is essentially the same, regardless of different intrinsic surface properties.     

The interaction between CD44 on tumour cells and hyaluronan under physiologic flow conditions: implications for metastasis formation

The adhesion of tumour cells to the endothelial cells of blood vessels of the microcirculation represents a crucial step in haematogenous metastasis formation. Similar to leukocyte extravasation, selectins mediate initial tumour cell rolling on endothelium. An additional mechanism of leukocyte adhesion to endothelial cells is mediated by hyaluronan (HA). However, data on the interaction of tumour cells with hyaluronan under shear stress are lacking. The expression of the hyaluronan binding protein CD44 on tumour cell surfaces was evaluated using flow cytometry. The adhesion of tumour cells to HA with regard to adhesive events and rolling velocity was determined in flow assays in the human small cell lung cancer (SCLC) cell lines SW2, H69, H82, OH1 and OH3, the colon carcinoma cell line HT29 and the melanoma cell line MeWo. Hyaluronan deposition in human and mouse lung blood vessels was histochemically determined. MeWo adhered best to HA followed by HT29. SCLC cell lines showed the lowest CD44 expression on the cell surface and lowest number of adhesive events. While hyaluronan was deposited in patches in the microvasculature of the alveolar septum in the human lung, it was only present in the periarterial space in the mouse lung. Certain tumour entities bind to HA under physiological shear stresses so that HA can be considered a further ligand for cell extravasation in haematogenous metastasis. As hyaluronan is deposited within the pulmonary microvasculature, it may well serve as a ligand for its binding partner CD44, which is expressed by many tumour cells.     

Effect of the hydrophobic surfactant proteins on the surface activity of spread films in the captive bubble surfactometer

The main function of pulmonary surfactant, a mixture of lipids and proteins, is to reduce the surface tension at the air/liquid interface of the lung. The hydrophobic surfactant proteins SP-B and SP-C are required for this process. When testing their activity in spread films in a captive bubble surfactometer, both SP-B and SP-C showed concentration dependence for lipid insertion as well as for lipid film refinement. Higher activity in DPPC refinement of the monolayer was observed for SP-B compared with SP-C. Further differences between both proteins were found, when subphase phospholipid vesicles, able to create a monolayer-attached lipid reservoir, were omitted. SP-C containing monolayers showed gradually increasing minimum surface tensions upon cycling, indicating that a lipid reservoir is required to prevent loss of material from the monolayer. Despite reversible cycling dynamics, SP-B containing monolayers failed to reach near-zero minimum surface tensions, indicating that the reservoir is required for stable films.