Adenovirus-mediated gene transfer of Lp-PLA

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 A 2 (Lp-PLA 2 ), an enzyme capable of hydrolyzing PAFlike phospholipids. In this study, we generated an adenovirus (Ad) encoding human Lp-PLA 2 and injected 10 8 , 10 9 , and 10 10 plaque-forming unit doses of Adlp-PLA 2 and control AdlacZ intra-arterially into rabbits to achieve overexpression of Lp-PLA 2 in liver and in vivo production of LpPLA 2 -enriched LDL. As a result, LDL particles with 3-fold increased Lp-PLA 2 activity were produced with the highest virus dose. Increased Lp-PLA 2 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-PLA 2 activity. Lipid accumulation and foam cell formation in RAW 264 macrophages were also decreased when incubated with oxidized LDL containing the highest Lp-PLA 2 activity. Inhibition of the Lp-PLA 2 activity in the LDL particles led to an increase in lipid accumulation and foam cell formation. It is concluded that increased Lp-PLA 2 activity in LDL attenuates foam cell formation and decreases LDL oxidation and subsequent degradation in macrophages. —Turunen, P., J. Jalkanen, T. Heikura, H. Puhakka, J. Karppi, K. Nyyssönen, and S. Ylä-Herttuala. Adenovirus-mediated gene transfer of Lp-PLA 2 reduces LDL degradation and foam cell formation in vitro. J. Lipid Res. 2004. 45: 1633–1639. Supplementary key words lipoprotein-associated phospholipase A 2 • low density lipoprotein • macrophage Oxidized low density lipoprotein (oxLDL) plays an important role in atherosclerosis. Uptake of oxLDL contributes to the formation of foam cells by arterial macrophages. OxLDL also plays other roles in atherogenesis, such as being cytotoxic and stimulating the migration of monocytes into the arterial wall (1). Oxidation of LDL is a free radical-mediated, autocatalytic process. Polyunsaturated fatty acids, which are present in LDL lipids, are main targets of the reactive forms of oxygen. Initial products of phospholipid oxidation are usually hydroperoxy derivatives, which give rise to a variety of aldehyde products (2). This fragmentation also 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 A 2 (Lp-PLA 2 ), also known as platelet-activating factor-acetylhydrolase (PAF-AH), which hydrolyzes them to lysophospholipids (4). Some of the biologic effects of oxLDL can be mimicked by phospholipids that are subjected to oxidation in vitro. The ability of oxidized phospholipids to induce monocyte binding can be completely abolished by LpPLA 2 (5). Atherosclerosis has features of chronic inflammation, and oxLDL has been implicated as a factor involved in arterial wall inflammation (6). During LDL oxidation, biologically active PAF-like phospholipid derivatives are produced that may contribute to the proinflammatory properties of oxLDL (7). PAF levels are found to be higher in coronary arteries from patients with severe atherosclerosis, and antibodies to PAF are associated with borderline hypertension, early atherosclerosis, and metabolic syndrome (8–10). Because it is difficult to increase Lp-PLA 2 activity in LDL particles in vitro without the risk of causing alterations in lipoprotein structure and physiological properties, we generated adenoviruses encoding Lp-PLA 2 cDNA and injected these viruses intra-arterially into rabbits to achieve overexpression of Lp-PLA 2 in liver and in vivo production of LDL particles with increased Lp-PLA 2 activity to study the effects of Lp-PLA 2 on LDL Abbreviations: Ad, adenovirus; ALAT, alanine aminotransferase; CRP, C-reactive protein; IL-6, interleukin-6; LPDS, lipoprotein-deficient serum; Lp-PLA 2 , lipoprotein-associated phospholipase A 2 ; lysoPC, lysophosphatidylcholine; oxLDL, oxidized low density lipoprotein; PAF-AH, platelet-activating factor-acetylhydrolase; pfu, plaque-forming units; SMC, smooth muscle cell; TBARS, thiobarbituric acid-reactive substance. 1 To whom correspondence should be addressed. e-mail: seppo.ylaherttuala@uku.fi Manuscript received 6 May 2004 and in revised form 23 June 2004. Published, JLR Papers in Press, July 1, 2004. DOI 10.1194/jlr.M400176-JLR200 by gest, on July 9, 2018 w w w . j.org D ow nladed fom