6.1. Enzymatically degraded LDL (E-LDL)
After infiltration into atherosclerotic lesions, LDL particles are degraded in situ by enzymes secreted by a variety of vascular cells, including macrophages. Enzymatic degradation of LDL in vitro is achieved by trypsin which degrades apoB, rendering underlying lipids accessible to cholesterol esterase for the cleavage of CE. This modified LDL particles are in structure, biological properties, and composition similar to lipid particles that accumulate in atherosclerotic lesions (147) and possess an atherogenic moiety (148). E-LDL is rapidly internalized by macrophages by means of a scavenger receptor (SR)-dependent pathway that is mediated partly via CD36 but not macrophage scavenger receptor A (MSR-A). (149).
Highest E-LDL binding was observed on CD14 high CD16+ (MNP2) monocytes, suggesting a selective interaction of E-LDL with distinct subpopulations of monocytes (149). In addition it could be demonstrated that FcgRIIA/CD32 has the highest surface expression density on MNP2, proposing an involvement of FcgRII/CD32 in E-LDL binding on peripheral blood monocyte subpopulations (149). Studies have demonstrated the binding of E-LDL to C-reactive protein (CRP) and complement activation (150). CRP is an acute-phase protein in humans which activates the complement system and is probably involved in early atherogenesis. On monocytes, specific CRP binding occurs through FcgRI/CD64 with low affinity as well as FcgRIIA/CD32 with high affinity (151). Immunohistochemical colocalization of E-LDL and activated complement in the extracellular space at the earliest stage of atherosclerotic lesion development (152) could be shown. Further selective adhesion and transmigration of monocytes is induced by E-LDL through endothelial cell monolayers (153).
Further E-LDL stimulates MCP-1 production in macrophages (154). Free fatty acids present in E-LDL selectively stimulate IL-8 secretion in endothelial cells which is crucial for firm adherence and transmigration of circulating monocytes into the intima (155). These data show that the extracellular generation of E-LDL by enzymatic degradation is an event occurring during the earliest stage of atherosclerosis and that E-LDL induces monocyte recruitment into atherosclerotic lesions and is internalized by monocyte-derived macrophages, leading to foam cell formation during atherogenesis.
6.2. Oxidized LDL (Ox-LDL)
A prerequisite for macrophage uptake and cellular accumulation of cholesterol is oxidative modification of LDL (156). The initiation of the oxidation process is induced by the intracellular generation of lipoperoxides which are transferred to LDL through the development of O2 derived free radicals. These species later initiate a series of chemical reactions that are generally referred to as lipid peroxidation. Ox-LDL, generated in vitro by treatment with copper as a catalyst and extensive dialysis, is taken up avidly by macrophages and can cause foamcell formation. Oxidation of LDL leads to a number of changes in the composition of the particle, which vary depending on the type and concentration of oxidant used and the time of exposure (157;158). The rates at which specific components undergo oxidation vary greatly. In general, the unsaturated fatty acyl chains of phospholipids, CE, and triglycerides are oxidized most readily, while cholesterol and saturated fatty acids react more slowly. In addition, a significant proportion of the unsaturated acyl chains of CE and phospholipids is oxidized to hydroperoxides, isoprostanes, and more advanced oxidation products (159). A small proportion of cholesterol is converted to oxysterols, initially 7-hydroperoxycholesterol. ApoB, the sole protein of LDL, is subject to both direct oxidative modification and reaction with products of lipid oxidation.
Depending on the degree of oxidation, in ‘minimally’, lightly or mildly Ox-LDL, apoB is modified only to a minor extent and is still able to bind to the LDL receptor and is taken up via clathrin coated pits while in heavily Ox-LDL, apoB prevent LDL from binding to LDL receptors, and instead Ox-LDL is recognised by receptors such as scavenger receptor A (SR-A) and CD36 (160;161).
Recent studies have indicated that the subsequent metabolism of Ox-LDL differs substantially from non-oxidized lipoproteins (162;163). In particular, Ox-LDL is poorly able to induce cytoplasmic CE accumulation. This appears to be the result of the chemical alterations during oxidation to components of LDL. For mildly oxidized LDL mediated accumulation, it is known that initially lysosomal CE hydrolysis generates free cholesterol accumulation in lysosomes (164). This cholesterol, however, is restricted from exiting the lysosome. This would suggest that certain modifications of lipoproteins, or excess free cholesterol itself, can inhibit trafficking of cholesterol out of the lysosome.
Mildly oxidized LDL also has a number of biological activities that are potentially proatherogenic. These include effects on macrophage viability, migration, and proliferation, and on cholesterol export, signalling, protein expression, and secretion. While modest levels of OxLDL stimulate macrophage survival and proliferation, there is agreement that high doses cause death. The toxic components of Ox-LDL are not well defined, but may include oxysterols (165). Ox-LDL contains lysophosphatidylcholine (LPC) which is a potent chemoattractant for macrophages (13) because it upregulates the expression of vascular cell
adhesion molecule such as ICAM-1 which is present within the endothelium and increases monocyte adhesion.
Ox-LDL itself is directly chemotactic for monocytes and T cells (but not for B cells or neutrophils, neither of which are found in lesions) (20;166). Among other biological effects, Ox-LDL (and its various oxidized lipid components) is cytotoxic for endothelial cells (167), mitogenic for macrophages and smooth muscle cells and stimulate the release of MCP-1 and of monocyte colony-stimulating factor (MCSF) from endothelial cells. Further, Ox-LDL can stimulate the production of many inflammatory mediators (e.g. endothelin-1) from other vascular cells, in turn resulting in diverse inflammatory responses in the arterial wall.
All atherogenic lipoproteins, once deposited in the intima, may exert direct or indirect proinflammatory effects. Table 2 summarizes the potential roles of Ox-LDL in atherogenesis, with special reference to its properties as an inflammatory mediator.
In summary atherogenic lipoproteins exert diverse effects by inducing chronic inflammatory reactions during lesion formation. They can augment the production of cytokines by vascular cells, and through the autocrine and paracrine mechanisms, the inflammatory reaction may lead to a vicious cycle resulting in lesion progression.
Table 2: Possible proatherogenic effects of oxidized LDL
Effects Possible mechanisms
Adhesion of monocytes to endothelial cells - Increased expression of adhesion molecules on endothelial cells
Monocyte and T lymphocyte chemotaxis - Induction of MCP-1 production and direct chemoattractant effect
Scavenger receptor A and CD36 - Activation of AP-1 and its transcription factors
Foam cell formation - Enhanced uptake of Ox-LDL mediated by scavenger receptors
Induction of proinflammatory genes Activation of NFkB, and AP-1, and increased cAMP
Increased cellular death Activation of apoptosis and formation of cholesterol crystals
Thrombosis - Induction of tissue factor, increased platelet aggregation
Impaired vascular functions Dysfunction of ET-1 and NO
Plaque rupture - Increased MMPs production
-: enhanced
Table from Jianglin Fan and Teruo Watanabe: inflammatory reactions in the pathogenesis of atherosclerosis, review. J Atheroscler Thromb, 2003; 10: 63-71 and see also Atherosclerosis an inflammatory disease, Schmitz G., Torzewski M, Inflammatory and Infectious Basis of Atherosclerosis, 2001, Birkhäuser Verlag, Basel/Switzerland.