Abstract-There are several well-documented functions of high-density lipoprotein (HDL) that may explain the ability of these lipoproteins to protect against atherosclerosis. The best recognized of these is the ability of HDL to promote the efflux of cholesterol from cells. This process may minimize the accumulation of foam cells in the artery wall. However, HDL has additional properties that may also be antiatherogenic. For example, HDL is an effective antioxidants. The major proteins of HDL, apoA-I and apoA-II, as well as other proteins such as paraoxonase that cotransport with HDL in plasma, are well-known to have antioxidant properties. As a consequence, HDL has the capacity to inhibit the oxidative modification of low-density lipoprotein (LDL) in a process that reduces the atherogenicity of these lipoproteins. HDL also possesses other antiinflammatory properties. By virtue of their ability to inhibit the expression of adhesion molecules in endothelial cells, they reduce the recruitment of blood monocytes into the artery wall. These antioxidant and antiinflammatory properties of HDL may be as important as its cholesterol efflux function in terms of protecting against the development of atherosclerosis.

Entry of monocytes into the vessel wall is an important event in atherogenesis. Previous studies from our laboratory suggest that oxidized arachidonic acid-containing phospholipids present in mildly oxidized low density lipoproteins (MM-LDL) can activate endothelial cells to bind monocytes. In this study, biologically active oxidized arachidonic acid-containing phospholipids were produced by autoxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (Ox-PAPC) and analyzed by liquid chromatography and electrospray ionization mass spectrometry in conjuction with biochemical derivatization techniques. We have now determined the molecular structure of two of three molecules present in MM-LDL and Ox-PAPC that induce monocyte-endothelial interactions. These lipids were identified as 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/z 594.3) and 1-palmitoyl-2-glutaryl-snglycero-3-phosphocholine (m/z 610.2). These two molecules were produced by unambiguous total synthesis and found to be identical by analytical techniques and bioactivity assays to those present in MM-LDL and Ox-PAPC. Evidence for the importance of all three oxidized phospholipids in vivo was suggested by their presence in fatty streak lesions from cholesterol-fed rabbits and by their immunoreactivity with natural antibodies present in ApoE null mice. Overall, these studies suggest that specific oxidized derivatives of arachidonic acidcontaining phospholipids may be important initiators of atherogenesis.

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-