Serum paraoxonase (PON1) is an esterase that is associated with high-density lipoproteins (HDLs) in the plasma; it is involved in the detoxification of organophosphate insecticides such as parathion and chlorpyrifos. PON1 may also confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids present in oxidized low-density lipoproteins (LDLs). To study the role of PON1 in vivo, we created PON1-knockout mice by gene targeting. Compared with their wild-type littermates, PON1-deficient mice were extremely sensitive to the toxic effects of chlorpyrifos oxon, the activated form of chlorpyrifos, and were more sensitive to chlorpyrifos itself. HDLs isolated from PON1-deficient mice were unable to prevent LDL oxidation in a co-cultured cell model of the artery wall, and both HDLs and LDLs isolated from PON1-knockout mice were more susceptible to oxidation by co-cultured cells than the lipoproteins from wild-type littermates. When fed on a high-fat, high-cholesterol diet, PON1-null mice were more susceptible to atherosclerosis than their wild-type littermates.

IntroductionOur group has previously demonstrated that oxidized phospholipids in mildly oxidized LDL (MM-LDL) Oxidation of LDL appears to play an important role in the development and progression of the atherosclerotic lesion (1 -3). Artery wall cells can produce oxidative species via multiple pathways which "seed" LDL trapped in the subendothelial space and initiate lipid oxidation (4-6). The biological properties of oxidized LDL in vitro appear to be dependent on the degree to which the lipid and protein components are oxidized.Mildly oxidized LDL (MM-LDL)' induces the activation of the nuclear transcription factor NFKB via a cAMP-dependent mechanism (7) and consequently induces endothelial cells to express monocyte-specific chemoattractants (8, 9), adhesion molecules (10), and colony stimulating factors (11). Highly oxidized LDL (Ox-LDL) is cytotoxic to artery wall cells (12)(13)(14) and is taken up by the scavenger receptor on macrophages (15-17). In humans, plasma levels of HDL are inversely correlated with the risk of clinically significant coronary events ( 18,19). Moreover, in vitro experiments have demonstrated that HDL inhibits LDL modification (9,14,20,21). Despite convincing evidence for the presence of oxidized LDL in the blood vessel wall (22)(23)(24) and the antiatherogenic properties of HDL, the interaction between these two lipoprotein particles is not well understood.Human serum paraoxonase (PON) is a calcium-dependent HDL-associated ester hydrolase that catalyzes the hydrolysis of organophosphates, aromatic carboxylic acid esters, and carbamates (25). The initial measurement of its activity was performed using synthetic substrates, therefore, an interesting, and as yet unresolved question, is what role PON plays in metabolizing substrates formed in vivo. PON is tightly associated with apolipoprotein A-I in HDL and has the highest activity in the liver and blood (25). Serum PON levels vary widely between different animal species (26) and among humans (27). Individuals with familial hypercholesterolemia and insulin-dependent diabetes mellitus have significantly lower serum levels of PON than do control individuals (28). Low levels of HDL-associated esterases are also correlated with susceptibility to myocardial infarction, fish eye disease, and Tangier disease (29,30). HDLassociated PON has been reported to inhibit copper-induced lipid peroxide generation in LDL (31). Our laboratory has recently shown that platelet activating factor-acetylhydrolase (PAF-AH), a phospholipase A2 that hydrolyzes short chain acyl groups and longer chain aldehydes esterified to the sn-2 position 1. Abbreviations used in this paper: BHT, butylated hydroxytoluene; CM-LDL, coculture-modified LDL; m/z mass to charge ratio; MM-LDL, mildly oxidized LDL; Ox-PAPC, oxidized l-palmitoyl-2-arachi-

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Background-The inflammatory/antiinflammatory properties of HDL were compared with HDL cholesterol in 2 groups of patients and in age-and sex-matched control subjects. Methods and Results-Group 1 consisted of 26 patients not yet taking a statin who presented with coronary heart disease (CHD) or CHD equivalents by National Cholesterol Education Program Adult Treatment Panel III criteria studied before and 6 weeks after 40 mg/d of simvastatin. Group 2 consisted of 20 patients with documented CHD and HDL cholesterol Ն84 mg/dL. The inflammatory/antiinflammatory properties of HDL were determined by the ability of the subject's HDL to alter LDL-induced monocyte chemotactic activity (MCA) in a human artery wall coculture. Induction of MCA by a control LDL was determined in the absence or presence of the subject's HDL. Values in the absence of HDL were normalized to 1.0. Values Ͼ1.0 after the addition of HDL indicated proinflammatory HDL; values Ͻ1.0 indicated antiinflammatory HDL. Group 1 values before simvastatin were LDL cholesterol, 118Ϯ24 mg/dL; HDL cholesterol, 57Ϯ13 mg/dL; triglycerides, 125Ϯ64 mg/dL; and high-sensitivity C-reactive protein (hs-CRP), 1.7Ϯ1.9 mg/L; and MCA values were 1.38Ϯ0.91, compared with 0.38Ϯ0.14 for control subjects (Pϭ1.5ϫ10

For more than two decades, there has been continuing evidence of lipid oxidation playing a central role in atherogenesis. The oxidation hypothesis of atherogenesis has evolved to focus on specific proinflammatory oxidized phospholipids that result from the oxidation of LDL phospholipids containing arachidonic acid and that are recognized by the innate immune system in animals and humans. These oxidized phospholipids are largely generated by potent oxidants produced by the lipoxygenase and myeloperoxidase pathways. The failure of antioxidant vitamins to influence clinical outcomes may have many explanations, including the inability of vitamin E to prevent the formation of these oxidized phospholipids and other lipid oxidation products of the myeloperoxidase pathway. Preliminary data suggest that the oxidation hypothesis of atherogenesis and the reverse cholesterol transport hypothesis of atherogenesis may have a common biological basis. The levels of specific oxidized lipids in plasma and lipoproteins, the levels of antibodies to these lipids, and the inflammatory/antiinflammatory properties of HDL may be useful markers of susceptibility to atherogenesis. Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides may both promote a reduction in oxidized lipids and enhance reverse cholesterol transport and therefore may have therapeutic potential. (1) reported that the oxidation of LDL was injurious to artery wall cells and suggested that LDL oxidation may be important in atherogenesis. They also demonstrated that HDL inhibited the LDL-induced cytotoxicity (1). Over the ensuing two decades, this group elucidated the basis for these observations and established the important role of oxidized cholesterol products, especially cholesterol hydroperoxides (2). THE SEARCH FOR MECHANISMS OF LDL-INDUCEDFOAM CELL FORMATION Also in 1979, Goldstein et al. (3) reported that acetylated LDL but not native LDL was taken up by "scavenger receptors" instead of the LDL receptor, resulting in cholesteryl ester accumulation in macrophages characteristic of foam cells. Because acetylation was not known to occur, after the publication of this seminal paper there was a search for physiological ligands that would explain foam cell formation. Fogelman et al. (4) soon reported that malondialdehyde, an obligate product of the oxidation of arachidonic acid by the lipoxygenase pathways, could cause Schiff-base formation with the epsilon amino groups of apolipoprotein B (apoB) lysines in LDL. The altered lipoprotein was recognized by macrophage scavenger receptors, resulting in cholesteryl ester accumulation characteristic of foam cells. The next year, Steinberg and colleagues (5) demonstrated that cultured endothelial