The anti-inflammatory effect of paraoxonase 1 against oxidized lipids depends on its association with high density lipoproteins Soumaya Loued a , Maxim Isabelle a , Hicham Berrougui a , Abdelouahed Khalil a, b, ⁎ a Research Center on Aging, University of Sherbrooke, Sherbrooke, Quebec, Canada b Geriatric Service, Department of Medicine, University of Sherbrooke, Sherbrooke, Quebec, Canada abstract article info Article history: Received 1 March 2011 Accepted 11 October 2011 Keywords: PON1 oxLDL HDL Inflammation ICAM-1 Aim: The aims of this study were to investigate whether purified PON1 can reduce the pro-inflammatory ef- fect of oxidized phospholipids and whether the effect depended on its association with HDL. Main methods: Lipid peroxidation was induced by copper ions and was measured using the conjugated diene method. Lysophosphatidylcholine (lyso-PC) formation was measured by HPLC with evaporative light scatter- ing detection (ELSD) and ICAM-1 expression on Ea.hy926 endothelial cells was analyzed by flow cytometry. Key findings: Purified PON1 significantly inhibited copper-induced oxidation of LDL and HDL, causing a 60.5% and 77.7% decrease in conjugated diene formation, respectively. Incubating PON1 with oxLDL caused a signif- icant increase in lyso-PC levels, while oxHDL caused a significant decrease. PON1 (12.5 to 50 μg/mL) had a pro-inflammatory effect in the presence of oxLDL, increasing ICAM-1 levels in Ea.hy926 cells by 33.0% and 40.6% (p b 0.001) respectively, and had an anti-inflammatory effect in the presence of oxHDL, causing a 3- fold reduction in ICAM-1 levels. PON1 also caused a significant decrease in TNFα and purified lyso-PC- induced ICAM-1 expression. The results obtained with reconstituted HDL as well as LCAT and PAF-AH inhibitors suggested that the anti-inflammatory effect of PON1 against oxidized lipids is dependent on its association with HDL. Significance: Our results clearly showed that PON1 is involved in the anti-inflammatory effect of HDL and that the effect appears to depend on its association with HDL. © 2011 Elsevier Inc. All rights reserved. Introduction Epidemiological studies of human populations have revealed that the risk of atherosclerotic events is strongly but inversely related to HDL levels. HDL, which are present in the interstitial space of the ar- tery wall at a much higher concentrations than LDL, appears to play a protective role by mediating reverse cholesterol transport, inhibiting LDL oxidation, and protecting endothelial cells against inflammation (Durrington, 1982). The protective effect of HDL has been attributed, in part, to the action of paraoxonase 1 (PON1), a calcium-dependent esterase closely associated with apolipoprotein A–I-containing HDL (Mackness et al., 1999; Watson et al., 1995a). PON1 activity is inversely related to cardiovascular risks (Mackness et al., 1999). Shih et al. (Shih et al., 1998) eloquently showed that PON1 plays a role in the anti-atherogenic properties of HDL. HDL from PON1 knockout mice do not protect LDL against oxidation or reduce the amount or chemotactic activity of MCP-1, unlike HDL from wild-type mice (Shih et al., 1998). PON1 also boosts HDL- mediated reverse cholesterol transport by increasing HDL binding to the ABCA-1 transporter (Efrat and Aviram, 2008). However, a study by Teiber et al. raised doubts about the antioxidant effect of PON1 (Teiber et al., 2004) and suggested that this activity may be due to tergitol or another protein contaminant (Teiber et al., 2004). To clarify this issue, we purified PON1 in presence of emul- gen 911 rather than tergitol and dialyzed the purified PON1 exten- sively before testing its antioxidant activity (Jaouad et al., 2003, 2006). Our results showed that PON1 has a strong antioxidant effect and protects LDL against oxidation (Jaouad et al., 2003, 2006). Moreover, the incubation of PON1 with N-ethylmaleimide (NEM), which irreversibly blocks the free SH group of PON1, causes a signif- icant reduction in the antioxidant activity of PON1 (Jaouad et al., 2006). Other in vitro and in vivo studies have also shown that PON1 protects LDL against lipid peroxidation and that HDL from PON1-knockout mice lose their antioxidant and anti-inflammatory activities (De Keyzer et al., 2009; Jaouad et al., 2006). PON1 has been reported to inhibit MCP1 induction in endothelial cells, likely due to its antioxidant ability (Mackness et al., 2004). Marsillach et al. suggested that PON1 protects against liver inflam- mation mediated by MCP-1 while Watson et al. suggested that PON1 possesses phospholipase-A2-like activity that allows it to hy- drolyze oxidized phospholipids at the sn-2 position (Marsillach et al., 2009; Watson et al., 1995b). A number of studies have Life Sciences 90 (2012) 82–88 ⁎ Corresponding author at: Research Center on Aging, 1036 Belvedere South, Sher- brooke, QC, Canada J1H 4C4. Tel.: +1 819 829 7131; fax: +1 819 829 7141. E-mail address: a.khalil@USherbrooke.ca (A. Khalil). 0024-3205/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2011.10.018 Contents lists available at SciVerse ScienceDirect Life Sciences journal homepage: www.elsevier.com/locate/lifescie