An elevated level of cholesterol impairs self-assembly of pulmonary surfactant into a functional film

Abstract

In adult respiratory distress syndrome, the primary function of pulmonary surfactant to strongly reduce the surface tension of the air-alveolar interface is impaired, resulting in diminished lung compliance, a decreased lung volume, and severe hypoxemia. Dysfunction coincides with an increased level of cholesterol in surfactant which on its own or together with other factors causes surfactant failure. In the current study, we investigated by atomic force microscopy and Kelvin-probe force microscopy how the increased level of cholesterol disrupts the assembly of an efficient film. Functional surfactant films underwent a monolayer-bilayer conversion upon contraction and resulted in a film with lipid bilayer stacks, scattered over a lipid monolayer. Large stacks were at positive electrical potential, small stacks at negative potential with respect to the surrounding monolayer areas. Dysfunctional films formed only few stacks. The surface potential of the occasional stacks was also not different from the surrounding monolayer. Based on film topology and potential distribution, we propose a mechanism for formation of stacked bilayer patches whereby the helical surfactant-associated protein SP-C becomes inserted into the bilayers with defined polarity. We discuss the functional role of the stacks as mechanically reinforcing elements and how an elevated level of cholesterol inhibits the formation of the stacks. This offers a simple biophysical explanation for surfactant inhibition in adult respiratory distress syndrome and possible targets for treatment.

FIGURE 1

Area-surface tension isotherm of BLES upon sample collection. The solid black line denotes the isotherm. Upon contraction of the film, the surface tension first dropped steeply before flattening into a shoulder of the isotherm. At the inflection point, a first sample was collected (denoted by 1). The film transfer is visible as a short horizontal stretch in the isotherm. The film area was then further reduced until the surface tension started to drop more steeply again, at which point a second sample was collected (2). After transfer of the second sample, the film was reexpanded and a third sample collected (3). Dotted lines denote the likely progression of the isotherm in the absence of sample collection. The expected progression was deduced from an isotherm of this film acquired before collection of the samples.

FIGURE 2

The discrete levels of lipid bilayer patches on top of each other were identified in each AFM micrograph in the histogram of the area count over topographical height using ImageJ (Wayne Rasband, National Institutes of Health, Bethesda, MD). In this example, a maximum of nine discrete levels of bilayers were identified.

FIGURE 3

AFM micrograph in three-dimensional representation of surfactant containing 5% w/w cholesterol (5 μm × 5 μm). The film shows stacks of bilayers up to three layers high. Each layer is five nanometers high.

FIGURE 4

AFM topographies (20 μm × 20 μm) of BLES containing no cholesterol (A), 5% w/w cholesterol (B), and 20% w/w cholesterol (C). The films denoted “expanded” have been collected from the Langmuir trough after the film was spread and allowed to contract up to the onset of a shoulder in the area-surface tension isotherm (γ = 34 mN/m, see Fig. 1). Then, the film area was further reduced and another sample collected (denoted contracted; (γ = 26 mN/m)). The images in the right column have been acquired from films after a partial reexpansion (γ = 34 mN/m). Films containing no or 5% cholesterol formed stacks of lipid bilayer patches, attached to the monolayer. The bilayer patches reintegrated in the monolayer when the film was reexpanded. Note that the reexpanded films of panels A and B still show some bilayer stacks because the area was only partially reexpanded. Films with 20% cholesterol formed almost no lipid bilayer stacks and continuously collapsed during contraction (C).

FIGURE 5

(A) Overview of a film of BLES that contains no cholesterol. The topographical image (left, and cross section below) shows a pattern of monolayer and scattered multilayer regions. In the potential map (right, and cross section below) large stacks of bilayer patches are at a potential of up to 200 mV above the monolayer. The arrows in the topographical image and in the potential map point to a region, where the topographical height does not change but the potential shows two distinct levels. (B) BLES film at higher magnification. The outer perimeters of the bilayer stacks from the topological image (left) are overlaid in red in the potential map (right). The larger of the bilayer stacks are at positive potential with respect to the monolayer. Small stacks are up to ∼100 mV lower in the electrical potential than average.

FIGURE 6

(A) Overview of a film of BLES that contains 20% cholesterol by weight. Protrusions in the topographical image (left) are not different in the potential map (right). (B) A high-resolution map of the topography (left) and potential (right) of the film containing 20% cholesterol. The perimeter of the small topographical features were computed from the topographical image and overlaid in red on the potential map. The monolayer is strongly electrically structured with a dynamic range of up to 300 mV. The length scale of the electrical domains is ∼100 nm. The perimeters shown in the overlay are enclosing an area of larger topographical height (i.e., larger film thickness). In the potential map, these areas coincide with the regions at a more positive surface potential.

FIGURE 7

Mechanism explaining a positive surface potential on the large bilayer stacks. The helical span of SP-C is oriented in lipid monolayers with ∼70° to the normal of the interface (51). Upon film contraction, a first bilayer adjacent to the monolayer is formed toward the aqueous subphase. SP-C rotates to span the newly formed bilayer and remains anchored to the monolayer by its two palmitoyl groups near its N-terminal end. SP-C is known to span lipid bilayers (52). As a result, all SP-C molecules face with the N-terminus toward the air, resulting in a positive surface potential. A related mechanism (not shown) may occur for the small bilayer stacks that were found to be at negative electrical surface potential. This time, the bilayer stacks may form toward the air. With the highly hydrophilic N-terminus remaining immersed in the water phase, SP-C may now point with its C-terminus toward the air.