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which recognizes the apo B100 moiety on the particle. The LDL receptor is then downregulated in response to cellular cholesterol concentrations [1]. Due to this it is recognized that the native LDL and the LDL receptor cannot be responsible for the creation of foam cells. One avenue of thought is that the LDL is oxidatively modified in order for the aberrant uptake of the cholesterol to occur in sufficient quantities to lead to the development of the foam cell. This hypothesis was first articulated by Steinberg and coworkers [2] when they noted that oxidation of LDL impacts on the pathophysiology of atherosclerosis by facilitating recruitment of monocytes into the arterial intima, preventing resident macrophages exiting the intima, and accelerating uptake of LDL. This resulted in the formation of foam cells and the loss of endothelial integrity due to the cytotoxic nature of the oxidized LDL (Ox-LDL).

Macrophages scavenge oxidatively modified LDL through the scavenger receptors, which is the main route for the uptake and accumulation of cholesterol in the quantities required. When the blood concentration of LDL is high, the intimal LDL concentration also increases. In the intimal location, the LDL is brought into close proximity with endothelial cells, which causes the LDL to be minimally oxidized (MM-LDL). MM-LDL is oxidized but

not to the extent that the apo B100 molecule is changed [3] therefore the LDL receptor can still recognize MM-LDL. MM-LDL is a chemoattractant for

mononuclear phagocytes and encourages their proliferation indirectly through the production of monocyte chemotactic protein-1 (MCP-1) [4]. MMLDL also stimulates the production of monocyte colony-stimulating factor (M-CSF), which stimulates mononuclear phagocytes to differentiate into tissue macrophages, which are therefore prevented from rejoining the circulation [4]. The presence of the macrophages stimulates further oxidation of the LDL. The macrophages take up the now Ox-LDL in an unregulated manner through the scavenger pathway, causing cholesterol accumulation and eventually the creation of the foam cell.

What is still largely unclear is where the oxidation occurs. It is unlikely to be in the antioxidant-rich environment of the plasma, and more likely to be in the microenvironment of the intima of the arterial wall where the local concentration of antioxidants can be exhausted allowing oxidation to progress [5]. It is generally believed that it is in the intima that the MM-LDL could be further oxidized to Ox-LDL. However, recent studies have shown that the quantities of antioxidants, in particular coantioxidants that could reduce the tocopherol radical, are nearly as high in the intima and in the atherosclerotic lesions as found in plasma [3].

In vitro the LDL can become modified in a number of ways, including enzymatically (principally through myeloperoxidase and 15-lipoxygenase) and through oxidation and due to this modification is retained in the arterial wall. These biologically active compounds stimulate the endothelium and attracted macrophages, T cells, mast cells, and smooth muscle cells to the fatty streak, leading to further oxidation of LDL [6]. This leads to the creation

of the fatty streak, which develops into a lesion and eventually atherosclerosis. The Ox-LDL hypothesis requires that oxidation is integral to the process of initiating and developing atherosclerosis and that antioxidants will slow or prevent the development of the atheroma [6].

The Evidence: for and against

There is evidence for the existence of the MM-LDL particle. Berliner et al. [4] have identified a particle in plasma that responds in a way that is consistent with a partially Ox-LDL molecule. Avagaro et al. [7] also identified subfractions of LDL that exhibit characteristics of Ox-LDL but are also sufficiently similar to native LDL.

A number of modifications occur to the LDL when it is oxidized by incubation, for example, with endothelial or smooth muscle cells. There is increased electrophoretic mobility toward the anode and extensive fragmentation occurs leading to the production of shortand long-chain aldehydes such as malondialdehyde (MDA) [8]. Some of these fragments are free, however others become bonded to the apo B100 or other lipids causing conjugated dienes. The surface phosphatidylcholine are vulnerable to hydrolysation to lysophosphatidylcholine. The removal of oxidized fatty acids from the surface of the LDL may have a role in the rapid propagation of the oxidation within the LDL itself [8]. The oxidation of cholesterol components can also lead to the fragmentation of apo B100. This fragmentation is not caused by proteolysis, as proteolytic inhibitors do not prevent the fragmentation, though antioxidants have been shown to do so in animals [4, 8, 9].

In Vitro Studies

The work of Goldstein and Brown [1] supplied the first evidence that something other than native LDL and the LDL receptor are required for sufficient accumulation of lipid that lead to foam cell formation. Mouse peritoneal macrophages, incubated with LDL in vitro, did not accumulate lipids, which is evidence that the macrophages had few LDL receptors. To support this, patients with familial hypercholesterolemia and the Watanabe heritable hyperlipidemic (WHHL) rabbit both develop lipid lesions while having only limited amounts of functional LDL receptors. To further support the lack of LDL receptor involvement in the development of atherosclerosis, in situ hybridization techniques show little or no mRNA for the LDL receptor in macrophage-rich lipid lesion [10].

Goldstein and Brown [11] subsequently showed that the LDL could be modified, and initial modification was through acetylation, which resulted in the uptake by the scavenger receptor of macrophages. In acetylation the ε-amino groups of the lysine residues are affected which causes a neutralization

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of the positive charge on the protein. This enables recognition by the scavenger receptor on the macrophage. Studies have shown that many such modifications are possible though many are also not likely under physiological conditions [8]. Oxidation of the LDL is a modification that is likely and also prevents recognition by the LDL receptor. It is likely that the lipids of the LDL themselves become oxidized, leading to the generation of peroxides and smallto long-chain aldehydes, such as MDA, which could then modify the apo B100. This modification could cause the Ox-LDL to be recognized by the scavenger receptor.

The scavenger receptor has been shown to be ubiquitously expressed on macrophages of different origin, and on endothelial cells, cultured smooth muscle cells of the rabbit, and on fibroblasts [8]. Via et al. [12] have characterized the scavenger receptor from a murine macrophage cell line, and similar receptor characterizations have followed from a number of studies using a variety of cell types [8]. Although the exact nature of the receptor recognition site is unclear, it is clear that it occurs in the apoprotein moiety of Ox-LDL. Parthasarathy et al. [13] showed that delipidated and resolublized Ox-LDL protein was still recognized by the scavenger receptor and competed with Ox-LDL for uptake. Similarly considerable animal evidence shows that where there is no scavenger receptor, for example mice lacking scavenger receptor-A gene, there is resistance to the development of atherosclerosis [3].

The adhesion of leukocytes to the endothelium, via the vascular cell adhesion molecule-1 (VCAM-1), appears to be influenced by oxidation. Interestingly the genes for the MCP-1 and VCAM-1 appear to be regulated by the redox-sensitive transcription factor NFκβ [14].

It has been shown that LDL incubated with endothelial cells becomes cytotoxic. Vascular cells and macrophages can produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) that Ox-LDL in vitro [3]. Henriksen et al. [15] showed that the modifications to the LDL, caused by proximity to the endothelial cells, resulted in uptake by the scavenger receptor, and was competitive with acetyl-LDL for uptake, which indicated a role for the same receptor in both modifications. The oxidative modification of LDL has been shown to act to promote atherosclerosis [6]. However, there is also evidence of another receptor that is not subject to competition by acetylLDL [16]. There are two forms of scavenger receptors A1 and A2. In addition to this, other receptors such as CD68, CD36, SR-B1, and LOX-1 also act in ways similar to the classic scavenger receptor [3].

Ox-LDL has been shown to have components that are powerful chemoattractants thought to be monocyte-specific, largely because neutrophils are rarely seen in atherosclerotic aorta [4]. This chemotactic activity is associated with the lysophosphatidylcholine (lyso PtdCho) that is generated during the oxidative modification of the LDL. The β-VLDL, isolated from cholesterolfed rabbit plasma, has high concentrations of lyso PtdCho and these β-VLDL are also potent chemotactic molecules for human monocytes [4].