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

Omega-6 (-6) polyunsaturated fatty acids (PUFA), abundant in the Western diet, are precursors for a number of key mediators of inflammation including the 2-series of prostaglandins (PG). PGE 2, a cyclooxygenase (COX) metabolite of arachidonic acid, a -6 PUFA, is a potent mediator of inflammation and cell proliferation. Dietary supplements rich in -3 PUFA reduce the concentrations of 2-series PG and increase the synthesis of 3-series PG (e.g., PGE 3 ), which are believed to be less inflammatory. However, studies on cellular consequences of increases in 3-series PG in comparison to 2-series PG have not been reported. In this study, we compared the effects of PGE 2 and PGE3 on (i) cell proliferation in NIH 3T3 fibroblasts, (ii) expression and transcriptional regulation of the COX-2 gene in NIH 3T3 fibroblasts, and (iii) the production of an inflammatory cytokine, IL-6, in RAW 264.7 macrophages. PGE 3, unlike PGE2, is not mitogenic to NIH 3T3 fibroblasts. PGE 2 and PGE3 both induce COX-2 mRNA via similar signaling mechanisms; however, compared with PGE 2, PGE3 is significantly less efficient in inducing COX-2 gene expression. Furthermore, although both PGE2 and PGE3 induce IL-6 synthesis in RAW 264.7 macrophages, PGE 3 is substantially less efficient compared with PGE2. We further show that increasing the -3 content of membrane phospholipid results in a decrease in mitogen-induced PGE2 synthesis. Taken together, our data suggest that successful replacement of -6 PUFA with -3 PUFA in cell membranes can result in a decreased cellular response to mitogenic and inflammatory stimuli.

The oxidation of apolipoprotein B-containing lipoproteins and cell membrane lipids is believed to play an integral role in the development of fatty streak lesions, an initial step in atherogenesis. We have previously shown that two antioxidant-like enzymes, paraoxonase (PON)-1 and PON3, are high density lipoprotein-associated proteins capable of preventing the oxidative modification of low density lipoprotein (LDL) (Reddy, S. T., Wadleigh, D. J., Grijalva, V., Ng, C., Hama, S., Gangopadhyay, A., Shih, D. M., Lusis, A. J., Navab, M., and Fogelman, A. M. (2001) Arterioscler. Thromb. Vasc. Biol. 21, 542-547). In the present study, we demonstrate that PON2 (i) is not associated with high density lipoprotein; (ii) has antioxidant properties; and (iii) prevents LDL lipid peroxidation, reverses the oxidation of mildly oxidized LDL (MM-LDL), and inhibits the ability of MM-LDL to induce monocyte chemotaxis. The PON2 protein was overexpressed in HeLa cells using the tetracyclineinducible ("Tet-On") system, and its antioxidant capacity was measured in a fluorometric assay. Cells that overexpressed PON2 showed significantly less intracellular oxidative stress following treatment with hydrogen peroxide or oxidized phospholipid. Moreover, cells that overexpressed PON2 were also less effective in oxidizing and modifying LDL and, in fact, were able to reverse the effects of preformed MM-LDL. Our results suggest that PON2 possesses antioxidant properties similar to those of PON1 and PON3. However, in contrast to PON1 and PON3, PON2 may exert its antioxidant functions at the cellular level, joining the host of intracellular antioxidant enzymes that protect cells from oxidative stress. Oxidation of low density lipoproteins (LDL)1 trapped in the arterial subendothelial space is a key process in atherosclerotic lesion development. High density lipoprotein (HDL) inhibits LDL oxidation (1-4) and prevents the synthesis and secretion of monocyte chemotactic protein-1 by artery wall cells, thereby blocking the recruitment and transmigration of monocytes though the arterial endothelial layer (5). The anti-atherogenic properties associated with HDL are due, at least in part, to the activity of HDL-associated enzymes, which interact with LDL and prevent and/or reverse its oxidation (6). The calcium-dependent ester hydrolase paraoxonase (PON)-1 (EC 3.1.8.1) is one of several such enzymes and is found tightly associated with apoA-I in the HDL particle (7). Purified PON1 not only prevents LDL oxidation (8), but also blocks the ability of mildly oxidized LDL (MM-LDL) to induce monocyte chemotaxis and binding to endothelial cells (9). Epidemiological studies have shown that PON1 polymorphisms are correlated with variations in plasma lipoprotein levels (10) and coronary artery disease in some populations (11-15). In addition, Mackness et al. (16) showed that alloenzymes of PON1 determine the effectiveness of HDL in the protection of LDL from lipid peroxidation. Two other members of the PON gene family, termed PON2 and PON3, have been identified...

The lipoprotein HDL has two important roles: first, it promotes reverse cholesterol transport, and second, it modulates inflammation. Epidemiological studies show that HDL-cholesterol levels are inversely correlated with the risk of cardiovascular events. However, many patients who experience a clinical event have normal, or even high, levels of HDL cholesterol. Measuring HDL-cholesterol levels provides information about the size of the HDL pool, but does not predict HDL composition or function. The main component of HDL, apolipoprotein A-I (apo A-I), is largely responsible for reverse cholesterol transport through the macrophage ATP-binding cassette transporter ABCA1. Apo A-I can be damaged by oxidative mechanisms, which render the protein less able to promote cholesterol efflux. HDL also contains a number of other proteins that are affected by the oxidative environment of the acute-phase response. Modification of the protein components of HDL can convert it from an anti-inflammatory to a proinflammatory particle. Small peptides that mimic some of the properties of apo A-I have been shown in preclinical models to improve HDL function and reduce atherosclerosis without altering HDL-cholesterol levels. Robust assays to evaluate the function of HDL are needed to supplement the measurement of HDL-cholesterol levels in the clinic.

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/ EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-B sites are not required for endotoxindependent induction. Inhibition of endotoxin-induced NF-B activation by expression of an inhibitor-B ␣ mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBP␤, and C/EBP␦ enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBP␤ represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.Macrophages play an important role in the regulation of inflammation and the immune response. When activated, macrophages release growth factors, cytokines, and lipid mediators such as prostaglandins and leukotrienes. Secreted prostaglandins promote inflammation by increasing vascular permeability (1) and vasodilation (2) and by directing cellular migration into the site of inflammation through the production and release of proinflammatory cytokines such as interleukin-6 (3). Induced prostaglandin synthesis is associated with the onset of symptoms resulting from acute immune system activation. For example, a knockout mouse strain unable to induce prostaglandin production does not develop fever in response to normally pyrogenic doses of bacterial endotoxin (4). Elevated prostaglandin levels are also associated with conditions of both chronic inflammation and cancer (5, 6). Because of the many potent effects of prostaglandins, control of prostaglandin synthesis is a critical element in the regulation of many physiological processes and the abatement of a number of pathophysiological conditions.The synthesis of prostaglandins is dependent on the activity of the cyclooxygenase (COX)

Background-These studies were designed to determine the mechanism of action of an oral apolipoprotein (apo) A-I mimetic peptide, D-4F, which previously was shown to dramatically reduce atherosclerosis in mice. Methods and Results-Twenty minutes after 500 g of D-4F was given orally to apoE-null mice, small cholesterolcontaining particles (CCPs) of 7 to 8 nm with pre-␤ mobility and enriched in apoA-I and paraoxonase activity were found in plasma.

Abstract-Paraoxonase-1 (PON1) is a secreted protein associated primarily with high density lipoprotein (HDL)