Adenovirus-mediated gene transfer of Lp-PLA2 reduces LDL degradation and foam cell formation in vitro

Hanna Puhakka,Tommi Heikura,Kristiina Nyyssönen,Päivi Turunen,Seppo Ylä-Herttuala,Johanna Jalkanen,Jouni Karppi

doi:10.1194/jlr.m400176-jlr200

Abstract

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity. These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAF-like phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA2 and injected 10(8), 10(9), and 10(10) plaque-forming unit doses of Adlp-PLA2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA2 in liver and in vivo production of Lp-PLA2-enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA2 activity were produced with the highest virus dose. Increased Lp-PLA2 activity in LDL particles decreased the degradation rate in RAW 264 macrophages after standard in vitro oxidation to 60-80% compared with LDL isolated from LacZ-transduced control rabbits. The decrease was proportional to the virus dose and Lp-PLA2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. Inhibition of the Lp-PLA2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages.

Highlights

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity
Initial products of phospholipid oxidation are usually hydroperoxy derivatives, which give rise to a variety of aldehyde products [2]. This fragmentation leads to the formation of polar phospholipids containing short-chain acyl groups at the sn-2 position [3]. These molecules serve as substrates for lipoprotein-associated phospholipase A2 (Lp-PLA2), known as platelet-activating factor-acetylhydrolase (PAF-AH), which hydrolyzes them to lysophospholipids [4]
Western blot analysis of lyophilized medium of the same cells showed in the Adlp-PLA2-transduced cell supernatants but not in the untransduced control cell supernatants the presence of an ‫ف‬65 kDa protein (Fig. 1B), which corresponds to the molecular mass identified for the glycosylated form of serum Lp-PLA2 [11]

Summary

Introduction

Oxidation of LDL generates biologically active platelet-activating factor (PAF)-like phospholipid derivatives, which have potent proinflammatory activity These products are inactivated by lipoprotein-associated phospholipase A2 (Lp-PLA2), an enzyme capable of hydrolyzing PAFlike phospholipids. Lipid accumulation and foam cell formation in RAW 264 macrophages were decreased when incubated with oxidized LDL containing the highest Lp-PLA2 activity. This fragmentation leads to the formation of polar phospholipids containing short-chain acyl groups at the sn-2 position [3] These molecules serve as substrates for lipoprotein-associated phospholipase A2 (Lp-PLA2), known as platelet-activating factor-acetylhydrolase (PAF-AH), which hydrolyzes them to lysophospholipids [4]. It was found that Lp-PLA2 gene transfer led to an increased enzyme activity in isolated LDL particles, with potentially antiatherogenic effects on LDL oxidation, subsequent degradation, and decreased foam cell formation in RAW 264 macrophages in vitro

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