Ox-LDL structure

Taken from: http://ethesis.helsinki.fi/

2.2.4. Oxidative modification of LDL

There is much evidence indicating that oxidized LDL (Ox-LDL) is present in atherosclerotic lesions in vivo (Ylä-Herttuala 1998). First of all, LDL isolated from atherosclerotic lesions is in part oxidatively modified (Ylä-Herttula et al. 1989). Second, immunological techniques have demonstrated that atherosclerotic lesions contain materials reactive with antibodies generated against Ox-LDL (Haberland et al. 1988, Palinski et al. 1989, Rosenfeld et al. 1990). Third, serum contains autoantibodies against Ox-LDL (Palinski et al. 1989, Salonen et al. 1992). Fourth, treatment with antioxidants can prevent the development or slow the progression of atherosclerosis (Carew et al. 1987, Kita et al. 1987, Steinberg 1997a). 

In principle, any modified LDL in plasma could be rapidly removed by hepatic sinusoidal cells (Kupffer cells), which contain abundant scavenger receptors. Moreover, a variety of antioxidants remain in plasma. Therefore, it was presumed that Ox-LDL mainly occurred locally in the arterial wall after entrance of normal LDL, whereby it was sequestered from antioxidants in plasma (Steinberg et al. 1989, Witztum and Steinberg 1991). However, recent studies have suggested that very small amounts of Ox-LDL are also present in plasma (see review from Nielsen 1999). These changes could have occurred elsewhere, or during a previous transient passage through the artery wall. Such minimally modified LDL might then be "primed" for more rapid oxidative modification on a subsequent entry into the intima. Therefore, Ox-LDL in the arterial wall can be derived both from normal LDL oxidized locally in the arterial intima and from Ox-LDL in plasma (Nielsen 1999). 

Lipid peroxidation presumably starts in the polyunsaturated fatty acids (PUFA) forming an ester bond with LDL-surface PLs, and then propagates to core lipids, resulting in oxidative modification not only of the PUFA, but also of the cholesterol moiety (mostly CE) and modification and degradation of apoB (Witztum 1994). 

Therefore, a wide variety of biologically active molecules can be formed, including oxidized sterols, oxidized fatty acids, and PL and protein derivatives generated by adduct formation with breakdown products of oxidized fatty acids. For example, malondialdehyde and 4-hydroxynonenal can subsequently react with lysine residues in apoB. Such adducts, and others, presumably create the epitopes on apoB that lead to recognition by scavenger receptors on macrophages.

In culture, all the vascular cells can initiate oxidation of LDL, but the relative contributions of ECs, monocytes and macrophages, or smooth muscle cells (SMC) to such modification in vivo are unknown (Heinecke 1998, Ylä-Herttuala 1998). In vitro, LDL can bind to copper which can promote rapid lipid peroxidation. However, it is not known whether sufficient free copper and iron, or complexes of these metals, exist in vivo to promote LDL peroxidation, although intact ceruloplasmin can act as a prooxidant. 

Therefore, several mechanisms are probably involved, and even the same cell type may use different pathways. For example, release of superoxide anion from ECs or SMCs might be responsible for initiation of oxidation in some settings, and thiols in others (Heinecke et al. 1986). 

In macrophages, enhanced 15-lipoxygenase activity could generate increased cellular lipid hydroperoxides, which could be transferred to extracellular LDL, providing the "seed" that would lead to enhanced lipid peroxidation (Heinecke 1998). 

The antioxidant defences that prevent oxidation of LDL need to be defined. The antioxidant content of the LDL particle is critical for its protection (Esterbauer et al. 1992) and, theoretically, if sufficient lipophilic antioxidants were present, the LDL particles would be protected from even profound oxidant challenge. In vivo, whether or not LDL becomes oxidized is a question of the balance between the extent of the prooxidant challenge and the capacity of the antioxidant defenses. 

Although Ox-LDL is found in man, there are no conclusive intervention studies in man to support a quantitatively important role for this process. The ongoing antioxidant trials will no doubt add more beneficial evidence in the role of atherosclerosis prevention. 

Antioxidants in LDL

Taken from: http://ethesis.helsinki.fi/

2.2.9. Antioxidants in the pathogenesis of atherosclerosis

As discussed in section 2.2.7, Ox-LDL is involved in several steps of atherosclerosis. It is believed that antioxidants can interfere to different extents with these steps (Faggiotto et al. 1998). Kleinveld et al. (1993) reported that 18 weeks of pravastatin or simvastatin administered to 23 hypercholesterolemic patients (15 men, 8 women) decreased LDL cholesterol levels by 36% and significantly reduced the rate and extent of copper-catalyzed LDL oxidation. LDL particles after therapy were changed in composition to contain less lipid relative to protein, possibly rendering the particle less susceptible to oxidation (Lavy et al. 1991). HMG-CoA reductase inhibitors rather than reducing LDL levels and changing its particle composition, may also be of antioxidant importance. In this regard, Giroux et al. (1993) reported that simvastatin diminished superoxide anion formation and LDL oxidation by human macrophages in tissue culture. Fruebis et al. (1994) have found that atherosclerosis in WHHL rabbits is inhibited by probucol (a potent antioxidant) but not by its analogue with similar structure. Thus, for any given prooxidant stress, there may be a threshold of protection that must be achieved. Protection of LDL from oxidation could increase nitric oxide bioavailability and improve endothelium-dependent vasomotor, anti-inflammatory, and anticoagulant properties of the endothelium (Guetta and Cannon 1996). Recently, Yasunari et al. (1999) observed that antioxidants probucol and vitamin E prevent smooth muscle cell migration and proliferation via reducing intracellular oxidative stress. Since endogenous antioxidants (superoxide dismutase, H₂O₂-removing enzymes, and metal binding proteins) may become inadequate to prevent LDL oxidation, exogenous antioxidants (diet-derived or supplemented) would seem, therefore, important to maintain such effect in vivo. The direct provision of lipophilic antioxidants into the LDL particle should be the most effective strategy. The most abundant natural antioxidant in LDL is a-tocopherol (Esterbauer et al. 1992), and supplementation of the diet with vitamin E can increase the vitamin E content of LDL and lead to enhanced protection of such LDL from in vitro oxidation. The vitamin E content in LDL particles is positively correlated with oxidation resistance (Tesoriere et al. 1998). However, in man, supplementation at about 1.2 g per day saturates the LDL, and at this degree of enrichment (about a 2 1/2-fold increase) the lag time for CD formation, a sensitive index of susceptibility to lipid peroxidation, is only prolonged by 50% (Reaven and Witztum 1996). Tikkanen et al. (1998) have observed that soybean phytoestrogen intake prolonged the lag time by 20 min. However, we do not know if this degree of lag time prolongation is sufficient to protect LDL under all conditions and its correlation to the prevention of CAD. For example, in hypercholesterolaemia, the residence of LDL in the artery is prolonged. If the pro-oxidant stress is continuous, more potent antioxidant activity would be required. Beta-carotene is the next most common antioxidant in LDL (Esterbauer et al. 1992) and theoretically should provide enhanced antioxidant protection. However, data have not supported this effect even when the b-carotene content was increased more than 20-fold by dietary enrichment (Heinecke 1998, Anderson et al. 1998). By contrast, the administration of probucol to volunteers, such that the probucol content of the LDL is 2-4 mg/mg of LDL protein, can lead to near-total protection against oxidative stress for as long as 16 hours. Vitamin C, a water-soluble antioxidant, also provides significant protection for LDL in vitro, presumably by maintaining or regenerating vitamin E in the LDL particle in its reduced antioxidant state.

Another strategy to protect LDL against oxidation is to reduce its content of PUFA by dietary substitution with oleic acid (Reaven et al. 1993). The diet-enriched flavonoids and isoflavonoids, may have great nutritional benefits against atherosclerosis as they appear to constitute a major source of dietary antioxidants (Hertog et al. 1993).

A reduction in prooxidant activity can also be achieved by enhancing the antioxidant content of cells, for example, by enriching them with ascorbate, or with vitamin E or b-carotene. Navab et al (1991) have developed a co-culture of ECs and SMCs that can oxidatively modify LDL. Enrichment of this culture with vitamin E, or b-carotene, decreases the ability of the cells to modify LDL.

In theory, antioxidant protection could be achieved with changes in lifestyle, diet and even with pharmacological approach. Despite the impressive ability of lipid-soluble antioxidants to block atherosclerosis in hypercholesterolemic animals, some studies are controversial because these antioxidants may have other antiatherogenic effects such as a hypolipidemic effect which may confound the conclusion. However, randomized clinical trials will eventually resolve the question as to whether these antioxidants deserve greater importance in inhibition of atherosclerosis.