Abstract
The global aim of our studies was to obtain more information about the mechanisms involved in the action of the hydrophobic surfactant components, with a special attention for SP-B. To reach this goal, many different assays and devices were used, including a pressure driven captive bubble surfactometer (CBS), a spreading
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trough and an atomic force microscope. The thesis starts with a general overview about the function and structural variation of the surfactant proteins (chapter 1). It is described that the activity and structure of the hydrophobic surfactant proteins SP-B and SP-C is very conserved among species throughout evolution, while more variation exists for that of SP-A and SP-D.
Aim i) of the thesis research was to obtain knowledge about the effect of SP-B and SP-C on the surface activity of spread films. This study, described in chapter 2, was done using the CBS, an apparatus that allows accurate determination of surface tension under dynamic conditions [24,25,26]. Surfactant components were spread at the air/liquid interface of an air-bubble, which was subsequently repeatedly compressed and expanded, thereby mimicking the breathing cycle. In this way information can be obtained on the role of individual surfactant components. Both SP-B and SP-C activity depended on the concentration of the proteins. Differences in surface dynamics between both proteins were found when lipid vesicles were omitted from the subphase.
Aim ii) was to investigate the role of cholesterol in surfactant. The effect of cholesterol on surface tension was examined by CBS, using spread lipid films containing SP-B and/or SP-C (chapter 3). It was shown that a cholesterol content of 10 mol% is optimal for surfactant activity in films containing SP-B, but not in films containing both SP-B and SP-C, or SP-C alone. Furthermore, differences in network structures were seen between films containing and those lacking cholesterol, as visualized by AFM.
Aim iii) was to investigate which surfactant components are responsible for the formation of protrusions upon monolayer compression towards low surface tensions (described in chapter 4). The appearance of compressed films containing saturated phospholipids plus native bovine SP-B was visualized by AFM. These films had similar appearance as those containing synthetic peptides based on the 25 N-terminal amino acids of SP-B. Protrusion height of lipid films containing SP-B1-25 peptide, however, was lower than that found for films containing native SP-B. It was further shown that the number of protruded layers depends on whether the phospholipids contain an unsaturated fatty acyl chain, and on whether the unsaturated acyl chain is present in phosphatidylcholine or in phosphatidylglycerol.
Aim iv) was to label a surfactant component and determine its distribution in a model of ARDS. In chapter 5 a method to label SP-B covalently at specific sites with the fluorophore Bodipy FL CASE is described. Using an array of techniques, fluorescently labeled SP-B was characterized and subsequently compared to the native protein. For instance, in a glass spreading trough mimicking dichotomic lung anatomy labeled SP-B spread on top of an aqueous surface and lowered surface tension independent of trough geometry. Since Bodipylabeled SP-B was shown to have retained its biophysical activity, it was supplemented to bovine lung surfactant extract and this was administered to rats in a model of ARDS (chapter 6). In this way the distribution of exogenous surfactant during surfactant replacement therapy was determined at the alveolar level. It was found that the majority of administered surfactant reached underinflated (dystelectatic) as well as aerated (open) alveoli, but hardly spread into severely damaged and collapsed (atelectatic) alveoli. The thesis is concluded with a summarizing discussion.
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