Effects of phospholipase A2 and its products on structural stability of human LDL: relevance to formation of LDL-derived lipid droplets

Shobini Jayaraman,Donald L Gantz,Olga Gursky

doi:10.1194/jlr.m012567

Abstract

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. To determine the effects of FFA on these transitions, we hydrolyzed LDL by phospholipase A(2) (PLA(2)), removed FFA by albumin, and analyzed structural stability of the modified lipoproteins. Earlier, we showed that heating induces LDL remodeling, rupture, and coalescence into lipid droplets resembling those found in atherosclerotic lesions. Here, we report how FFA affect these transitions. Circular dichroism showed that mild LDL lipolysis induces partial β-sheet unfolding in apolipoprotein B. Electron microscopy, turbidity, and differential scanning calorimetry showed that mild lipolysis promotes LDL coalescence into lipid droplets. FFA removal by albumin restores LDL stability but not the protein conformation. Consequently, FFA enhance LDL coalescence into lipid droplets. Similar effects of FFA were observed in minimally oxidized LDL, in LDL enriched with exogenous FFA, and in HDL and VLDL. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.

Highlights

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation
To test whether lipoprotein coincubation at 37°C with phospholipase A2 (PLA2) led to proteolysis, lipoproteins hydrolyzed to stages 1–3 were subjected to SDS PAGE
The results reported here reveal that the heat-induced coalescence of LDL into lipid droplets is greatly enhanced upon FFA incorporation and is hampered upon FFA removal by albumin

Summary

Introduction

Hydrolysis and oxidation of LDL stimulate LDL entrapment in the arterial wall and promote inflammation and atherosclerosis via various mechanisms including lipoprotein fusion and lipid droplet formation. Our results imply that FFA promote lipoprotein coalescence into lipid droplets and explain why LDL oxidation enhances such coalescence in vivo but hampers it in vitro. Such lipid droplet formation potentially contributes to the pro-atherogenic effects of FFA.—Jayaraman, S., D. Hydrolytic and oxidative modifications can induce LDL aggregation, fusion, and coalescence into lipid droplets, which further enhance LDL retention in the arterial wall [6]. Because nonmodified LDL do not fuse under physiologic conditions, modifications such as oxidation, lipolysis, and proteolysis are thought to be prerequisites for lipoprotein fusion [(1–4, 10) and references therein] The effects of these modifications on LDL aggregation and fusion have been attributed to the packing defects in the particle surface [6, 8], which may result from an imbalance between this surface and the apolar core [12].

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