No abstract

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

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.

Abstract-Oxidation of low density lipoprotein (LDL) phospholipids containing arachidonic acid at the sn-2 position occurs when a critical concentration of "seeding molecules" derived from the lipoxygenase pathway is reached in LDL. When this critical concentration is reached, the nonenzymatic oxidation of LDL phospholipids produces a series of biologically active, oxidized phospholipids that mediate the cellular events seen in the developing fatty streak. Normal high density lipoprotein (HDL) contains at least 4 enzymes as well as apolipoproteins that can prevent the formation of the LDL-derived oxidized phospholipids or inactivate them after they are formed. In the sense that normal HDL can prevent the formation of or inactivate these inflammatory LDL-derived oxidized phospholipids, normal HDL is anti-inflammatory. HDL from mice that are genetically predisposed to diet-induced atherosclerosis became proinflammatory when the mice are fed an atherogenic diet, injected with LDL-derived oxidized phospholipids, or infected with influenza A virus. Mice that were genetically engineered to be hyperlipidemic on a chow diet and patients with coronary atherosclerosis, despite normal lipid levels, also had proinflammatory HDL. It is proposed that LDL-derived oxidized phospholipids and HDL may be part of a system of nonspecific innate immunity and that the detection of proinflammatory HDL may be a useful marker of susceptibility to atherosclerosis. (Arterioscler Thromb Vasc Biol. 2001;21:481-488.)Key Words: HDL Ⅲ LDL Ⅲ atherosclerosis Ⅲ oxidized phospholipids T he events involved in fatty streak formation resemble those elicited by mycobacteria. 1 Over the past decade, there has been increasing evidence that this inflammatory response may, in part, be elicited by the oxidation of phospholipids contained in LDL. 2,3 Several oxidized phospholipids that are able to induce the genes and proteins necessary for the cellular response seen in the fatty streak have been identified in mildly oxidized LDL and in lesions of animal models of atherosclerosis. 4 -9 Two of these oxidized phospholipids, 1-palmitoyl-2(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine (POVPC) and 1-palmitoyl-2-glutaroyl-snglycero-3-phosphorylcholine (PGPC), both induced monocytes to bind to endothelial cells. 10 However, PGPC but not POVPC also induced neutrophils to bind to endothelial cells. 10 Indeed, POVPC strongly inhibited lipopolysaccharide-mediated induction of neutrophil binding and expression of E-selectin protein and mRNA. 10 This inhibition by POVPC was mediated by a protein kinase A-dependent pathway that resulted in downregulation of nuclear factor-B-dependent transcription. 10 PGPC, on the other hand, induced both E-selectin and vascular cell adhesion molecule-1 (VCAM-1) expression on endothelial cells. 10 On the basis of studies in Xenopus laevis oocytes, Leitinger et al 10 concluded that POVPC and PGPC bound to different receptors. Furthermore, they demonstrated that at concentrations equal to those present in mildly oxidized LDL, POVPC prevente...

We have examined the effects of mildly oxidized LDL and atherosclerosis on the levels of two proteins associated with HDL; apolipoprotein J (apoJ), and paraoxonase (PON). On an atherogenic diet, PON activity decreased by 52%, and apoJ levels increased 2.8-fold in fatty streak susceptible mice, C57BL/6J (BL/6), but not in fatty streak resistant mice, C3H/HeJ (C3H). Plasma PON activity was also significantly decreased, and apoJ levels were markedly increased in apolipoprotein E knockout mice on the chow diet, resulting in a 9.2-fold increase in the apoJ/PON ratio as compared to controls. Furthermore, a dramatic increase in the apoJ/PON ratio (over 100-fold) was observed in LDL receptor knockout mice when they were fed a 0.15%-cholesterol-enriched diet. Injection of mildly oxidized LDL (but not native LDL) into BL/6 mice (but not in C3H mice) on a chow diet resulted in a 59% decrease in PON activity ( P Ͻ 0.01) and a 3.6-fold increase in apoJ levels ( P Ͻ 0.01). When an acute phase reaction was induced in rabbits, or the rabbits were placed on an atherogenic diet, hepatic mRNA for apoJ was increased by 2.7-fold and 2.8-fold, respectively. Treatment of HepG2 cells in culture with mildly oxidized LDL (but not native LDL) resulted in reduced mRNA levels for PON (3.0-fold decrease) and increased mRNA levels for apoJ (2.0-fold increase). In normolipidemic patients with angiographically documented coronary artery disease who did not have diabetes and were not on lipid-lowering medication ( n ϭ 14), the total cholesterol/HDL cholesterol ratio was 3.1 Ϯ 0.9 as compared to 2.9 Ϯ 0.4 in the controls ( n ϭ 19). This difference was not statistically significant. In contrast, the apoJ/PON ratio was 3.0 Ϯ 0.4 in the patients compared to 0.72 Ϯ 0.2 in the controls ( P Ͻ 0.009). In a subset of these normolipidemic patients ( n ϭ 5), the PON activity was low (48 Ϯ 6.6 versus 98 Ϯ 17 U/ml for controls; P Ͻ 0.009), despite similar normal HDL levels, and the HDL from these patients failed to protect against LDL oxidation in co-cultures of human artery wall cells. We conclude that: ( a ) mildly oxidized LDL can induce an increased apoJ/PON ratio, and ( b ) the apoJ/PON ratio may prove to be a better predictor of atherosclerosis than the total cholesterol/HDL cholesterol ratio. ( J. Clin. Invest.