Molecular-Scale Biophysical Modulation of an Endothelial Membrane by Oxidized Phospholipids

Abstract

The influence of two bioactive oxidized phospholipids on model bilayer properties, membrane packing, and endothelial cell biomechanics was investigated computationally and experimentally. The truncated tail phospholipids, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), are two major oxidation products of the unsaturated phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-phosphocholine. A combination of coarse-grained molecular dynamics simulations, Laurdan multiphoton imaging, and atomic force microscopy microindentation experiments was used to determine the impact of POVPC and PGPC on the structure of a multicomponent phospholipid bilayer and to assess the consequences of their incorporation on membrane packing and endothelial cell stiffness. Molecular simulations predicted differential bilayer perturbation effects of the two oxidized phospholipids based on the chemical identities of their truncated tails, including decreased bilayer packing, decreased bilayer bending modulus, and increased water penetration. Disruption of lipid order was consistent with Laurdan imaging results indicating that POVPC and PGPC decrease the lipid packing of both ordered and disordered membrane domains. Computational predictions of a larger membrane perturbation effect by PGPC correspond to greater stiffness of PGPC-treated endothelial cells observed by measuring cellular elastic moduli using atomic force microscopy. Our results suggest that disruptions in membrane structure by oxidized phospholipids play a role in the regulation of overall endothelial cell stiffness.

Figure 1

OxPC structure and simulation systems. (A) Chemical structures and CG topologies of oxidized lipid molecules POVPC (left) and PGPC (right). Polar headgroups and glycerol backbones are purple spheres, sn-1 tails are blue spheres, and sn-2 truncated oxidized tails are red spheres circled by blue-dashed lines. (B) Snapshot of the simulation box containing water (cyan solvent representation), Na⁺ counterions (brown spheres), and a multicomponent bilayer containing 10 mol % PGPC. (C) Simulation snapshots of the equilibrated bare bilayer (left) and bilayers containing 10 mol % POVPC (center) and 10 mol % PGPC (right). POPC is depicted with the headgroup and backbone as white spheres and the tails as yellow spheres, DPPC as gray spheres and SM as pink spheres. The oxidized lipids (center and right) are represented with colors as described in (A) and with the remaining lipids transparent. To see this figure in color, go online.

Figure 2

Computational analysis of lipid order in POVPC- and PGPC-containing bilayers. (A) Probability distributions of tail tilt angles for POPC (solid black lines), POVPC (dashed red lines), and PGPC (dotted blue lines). POPC distributions are shown to indicate the usual positions of phospholipid tails. (B) Orientational order parameters (P in Eq. 6) of phospholipid tail pseudobonds for bare bilayers (gray squares) and bilayers containing POVPC (red circles) and PGPC (blue triangles). (C) CG structures of each phospholipid are depicted with tails colored differently than headgroups and glycerol backbones. Bond numbers from (B) are indexed for the DPPC structure (top). (D) Order parameters for the third pseudobond of each phospholipid tail. (Data are shown as time- and ensemble-averaged mean ± SE for n = 63–280 lipid molecules for each bilayer; ^∗p < 0.01). To see this figure in color, go online.

Figure 3

Computational analysis of water penetration into POVPC- and PGPC-containing bilayers. (A) Simulation snapshots of water penetration into bilayers. CG water particles are cyan spheres. Approximate positions of lipid phosphate groups in each leaflet are indicated by arrows. (B) Water-density profiles for one leaflet of bare bilayer (solid black line) and bilayers containing POVPC (dashed red line) and PGPC (dotted blue line). The bilayer center is located at 0 Å and the glycerol backbone at ∼16 Å. (Data are shown as the mean ± SE for n = 250 simulation snapshots; ^∗p < 0.01). (C) Water content of the PGPC bilayer normalized as a percentage of the bare bilayer water content. (Data shown as the mean ± SEM; n = 250 simulation snapshots. ^∗p < 0.01). (D) Water content of the PGPC bilayer separated into headgroup, glycerol backbone, and tail regions and compared to equivalent regions in the bare bilayer. (Data are shown as the mean ± SE for n = 250 simulation snapshots; ^∗p < 0.01). To see this figure in color, go online.

Figure 4

Impact of oxPCs on the bending moduli of bilayers. (A) Undulation spectra in the small-q regime versus the wave number (q) fitted to theoretical q⁻⁴ lines (Eq. 1) for the bare (black squares), POVPC (red circles), and PGPC (blue triangles) systems obtained over 800 ns of simulation time. Open symbols represent block averages of the intensity at each wave-number value, with error bars indicating the mean ± SE. Linear fits to each data set are shown in corresponding colors. The q⁻⁴ line is overlaid as a thicker dashed line. (Inset) Snapshot of the simulation box containing an undulating bare bilayer system consisting of 2500 lipids. All lipids are shown with white headgroups and glycerol backbones and yellow tails. The phosphate groups used to obtain undulation spectra are shown as larger mauve spheres. Water has been hidden for clarity. (B) Bending modulus estimates for the bare, POVPC, and PGPC bilayers, obtained from the intercepts of the fit lines in (A) with the vertical axis. Data were obtained by block averaging 100 ns simulation sections and are shown as the mean ± SEM for n = 8 blocks, each containing 500 simulation snapshots; ^∗p < 0.01. To see this figure in color, go online.

Figure 5

POVPC and PGPC disrupt lipid packing in endothelial membranes. (A) Typical GP images of control and POVPC- and PGPC-treated cells. (B) GP histograms for three experimental cell populations (navy squares) fitted by two-Gaussian distributions, with the right-shifted curve (yellow) representing ordered domains and the left-shifted curve (blue) representing fluid domains. The sum of the Gaussians is shown as a solid mauve curve. The GP distribution is obtained from the region −0.35 to + 0.8. (C) Average GP values were obtained from peaks of the fitted Gaussian distributions for the ordered and disordered domains (n = 12–25 images/experiment for three independent experiments; ^∗p < 0.05). To see this figure in color, go online.

Figure 6

OxPAPC, POVPC, and PGPC induce endothelial cell stiffening, which is reversible by addition of cholesterol. (A) Histogram of elastic moduli measured in endothelial cells treated with PAPC, oxPAPC, and oxPAPC with MβCD-cholesterol (oxPAPC Cholesterol). (B) Histograms of elastic moduli measured in control cells (ethanol only) and cells treated with POVPC and PGPC. (C) Average elastic moduli in cells treated with PAPC, oxPAPC, and oxPAPC with MβCD-cholesterol (oxPAPC Chol). (D) Average elastic moduli of control cells and cells under POVPC and PGPC conditions (n = 40–50 cells; ^∗p < 0.05).