Ox-LDL

Exposure of LDL to free radicals leads to lipid peroxidation and to a progressive loss of vitamin E and carotenoid within 6h. Thereafter the polyunsaturated fatty acids 18 2 and 20 4 are degraded in a lipid-peroxidation process [17] and a large variety of aldehydes is formed the following have been identified and quantified 4-hydroxyhexanal, 4-hydroxyoctenal, 4-hydroxynonenal, propanal, butanal, pentanal, hexanal, 2,4-heptadienal and malonaldehyde (MDA). 

Further, there is an extensive fragmentation of the apoB to smaller peptides which do not bind to the B/E receptor but to the scavenger receptors [20]. 

Oxidised LDL is toxic toward endothelial cells, smooth muscle cells and fibroblasts [18]. The toxic principle has not yet been identified, but it is proven that the protein moiety is not required. The toxic components reside in the lipid phase of Ox-LDL. In cultured fibroblasts and endothelial cells, the cytotoxicity and inhibition of growth essentially reside in 2,4-alkadienals (nonadienal, decadienal) and 4-hydroxynonenal, which produce 50% inhibition of endothelial cell proliferation. Lysophosphatides are present in Ox-LDL and have been shown to produce deleterious effects in cells, such as inhibition of the Na/K pump [18]. It has been suggested that oxidation of LDL results in an activation of a previously masked phospholipase A2 activity of apoB [21], which results in the release of lysophosphatides and oxidised fatty acids. Lysophosphatides act as chemoattractants for blood monocytes [22], 

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Fig. 2.1 Sequence of events in atherogenesis and role of low-density lipoprotein. Native LDL, in the subendothelial space, undergoes progressive oxidation (mmLDL) and activates the expression of MCP-1 and M-CSF in the endothelium (EC). MCP-1 and M-CSF promote the entry and maturation of monocytes to macrophages, which further oxidise LDL (oxLDL). Ox-LDL is specifically recognised by the scavenger receptor of macrophages and, once internalised, formation of foam cells occurs. Both mmLDL and oxLDL induce endothelial dysfunction, associated with changes of the adhesiveness to leukoc)des or platelets and to wall permeability.

Although atherosclerosis and rheumatoid arthritis (RA) are distinct disease states, both disorders are chronic inflammatory conditions and may have common mechanisms of disease perpetuation. At sites of inflammation, such as the arterial intima undergoing atherogen-esis or the rheumatoid joint, oxygen radicals, in the presence of transition-metal ions, may initiate the peroxidation of low-density lipoprotein (LDL) to produce oxidatively modified LDL (ox-LDL). Ox-LDL has several pro-inflammatory properties and may contribute to the formation of arterial lesions (Steinberg et /., 1989). Increased levels of lipid peroxidation products have been detected in inflammatory synovial fluid (Rowley et /., 1984 Winyard et al., 1987a Merry et al., 1991 Selley et al., 1992 detailed below), but the potential pro-inflammatory role of ox-LDL in the rheumatoid joint has not been considered. We hypothesize that the oxidation of LDL within the inflamed rheumatoid joint plays a pro-inflammatory role just as ox-LDL has the identical capacity within the arterial intima in atherosclerosis. 

This chapter addresses (1) the mechanisms, antioxidant defences and consequences in relation to free-radical production in the inflamed rheumatoid joint (2) lipid abnormalities in RA (3) the potential contribution of ox-LDL to RA (the role of ox-LDL in coronary heart disease is discussed in Chapters 2 and 3 and will not be fully discussed here) and (4) the therapeutic aspects of chain-breaking antioxidant interventions in RA. 

In contrast to MDA and hydroxynonenai, other aldehyde products of lipid peroxidation are hydrophobic and remain closely associated with LDL to accumulate to mil-limolar concentrations. Aldehydes at these elevated levels react with the protein portion of the LDL molecule, apolipoprotein B (apoB). Accumulated aldehydes bind the free amino groups from lysine residues in addition to other functional groups (-OH, -SH) on the apoB polypeptide. Consequently, the protein takes on a net negative charge and complete structural rearrangement results in the formation of ox-LDL. ox-LDL is no longer recognized by the LDL receptor, and has several pro-inflammatory properties (discussed below). 

NF-xB activation has been linked with atherosclerosis (Andalibi etal., 1993 Liao etui., 1993). Mice that were maintained on an atherogenic diet, which resulted in ox-LDL accumulation in the liver and arteries, showed NF-xB activation in hepatic tissues. Furthermore, inflammatory gene up-regulation corresponded to the concentration of accumulated lipid peroxides as well as genetic susceptibility to fetty-streak development. 

It is unlikely that the damaging effects of ox-LDL are relevant only to the walls of blood vessels and there is no reason to suppose they are confined to one disease. The initial histopathologjcal sign of coronary heart disease is the appearance of the fetty streak on the luminal surfece of arteries. Fatty streaks are composed of aggregated macrophages that have taken up ox-LDL via the scavenger receptor. Recently, we have detected such foam cells in the rheumatoid synovium (Section 5.5). 

The LDL particle, which has been oxidatively modified by the mechanisms described above, is no longer recognized by the classic LDL receptor and is taken up by the macrophage scavenger receptor. Importantly, ox-LDL also exhibits a variety of pro-inflammatory activities, as described below. 

Rheumatoid synovial cells produce a broad range of cytokines (Brennan et al., 1991). The formation of IL-la, IL-1 /3, GM-CSF, G-CSF, TNFa and other cytokines may be under the direct influence of ox-LDL and/or... 

Oxidatively modified LDL up-regulates the surfece expression of VCAM-1 and intracellular adhesion molecule-1 (ICAM-1) in cultured endothelial cells, promoting the interactions between both cell types (Kume et al., 1992). This may play a pivotal role in the development of atherosclerosis by promoting the penetration of circulating monocytes into the suben-dothelial space whilst inhibiting the mobility of resident macrophages. It has been previously demonstrated that ICAM-1, E-selectin, and VCAM-1 are up-regulated in the microvasculature of rheumatoid but not control synovium (Corkill et al., 1991 Koch et al., 1991). The association between ox-LDL and increased expression of adhesion molecules in the inflamed synovium has yet to be studied. 

In atherosclerosis, ox-LDL is taken up ultimately by macrophages and smooth muscle cells in the arterial intima. Once loaded with lipid, these cells have a foamy appearance when examined histologically. The accumulation of these so-called foam cells in the artery wall leads to the formation of fatty streaks , which can lead to atheromatous plaque formation and consequent coronary heart disease. 

OH-Ade 8-hydroxyadenine 6-OHDA 6-hydroxyguanine 8-OH-dG 8-hydroxydeoxyguanosine also known as 7,8-dihydro-8-0X0-2 -deoxyguanosine 8-OH-Gua 8-hydroxyguanine OHNE Hydroxynonenal 4-OHNE 4-hydroxynonenal OT Oxytocin OVA Ovalbumin ox-LDL Oxidized low-density lipoprotein... 

Cominacini, L., Pasini, A.F., Garbin, U., DavoU, A., Tosetti, M.L., Campagnola, M., Rigoni, A., Pastorino, A.M., Lo Casdo, V., and Sawamura, T., 2000, Oxidized low density lipoprotein (ox-LDL) binding to ox-LDL receptor-1 in endothelial cells induces the activation of NF-kappaB through an increased production of intracellular reactive oxygen species, J. Biol. Chem. 275 12633-12638. 

Jovinge, S., Ares, M.P., Kallin, B., and Nilsson, J., 1996, Human monocytes/macrophages release TNF-alpha in response to Ox-LDL, Arterioscler. Thromb. Vase. Biol. 16 1537-1539. 

Li, D., Saldeen, T., Mehta, J.L., 1999, gamma-tocopherol decreases ox-LDL-mediated activation of nuclear factor-kappaB and apoptosis in human coronary artery endothelial cells. Biochem. Biophys. Res. Commun. 259 157-161. 

Pretreatment of neurons by flavonoids (epicatechin and its 3 -D-methylether, kaempferol) strongly inhibits cell death induced by oxidized low-density lipoproteins (ox-LDL) without reduction of ox-LDL uptake or intracellular oxidative stress. Cell protection is selectively correlated to inactivation of JNK, thus suggesting that, irrespective of their H-atom donating activity, flavonoids can selectively attenuate a pro-apoptotic signaling cascade involving MAPKs. 

Fig. (3). Mechanisms implicated in the protective effect of flavonoids in LDL oxidation. OX-LDL oxidized LDL CE cholesteryl ester UC unesterified cholesterol GSH glutathione SOD superoxide dismutase ROS radical oxygen species. Dashed lines represent inhibition.

From the studies of atherosclerosis, we know that oxidative stress and inflammatory processes are of major importance because they stimulate oxidized LDL (Ox-LDL) induced macrophage cholesterol accumulation... 

The effect of PJ consumption by patients with CAS on their serum oxidative state was measured also as serum concentration of antibodies against Ox-LDL.31 A significant (p < 0.01) reduction in the concentration of antibodies against Ox-LDL by 24 and 19% was observed after 1 and 3 months of PJ consumption, respectively (from 2070 61 EU/mL before treatment to 1563 69 and 1670 52 F.lI/mL after 1 and 3 months of PJ consumption, respectively). Total antioxidant status (TAS) in serum from these patients was substantially increased by 2.3-fold (from 0.95 0.12 nmol/L at baseline up to 2.20 0.25 nmol/L after 12 months of PJ consumption). These results indicate that PJ administration to patients with CAS substantially reduced their serum oxidative status and could thus inhibit plasma lipid peroxidation. The susceptibility of the patient s plasma to free radical-induced oxidation decreased after 12 months of PJ consumption by 62% (from 209 18 at baseline to 79 6 nmol of peroxides/milliliter). The effect of PJ consumption on serum oxidative state was recently measured also in patients with non-insulin-dependent diabetes mellitus (NIDDM). Consumption of 50 mL of PJ per day for a period of 3 months resulted in a significant reduction in serum lipid peroxides and thiobarbituric acid reactive substance (TBAR) levels by 56 and 28%, respectively.32... 

Figure 8.5 Pomegranate by-product (PBP) consumption by E° mice attenuates atherosclerotic lesion development, in association with reduction in macrophage oxidative stress and Ox-LDL uptake. E° mice consumed PBP (17 or 51.5 mg gallic acid equivalents/kilogram/day) for 3 months. Control mice received only water (placebo). At the end of the study, the mice aortas as well as the mice peritoneal macrophages were harvested. (A) Atherosclerotic lesion size determination. (B) Total macrophage peroxide levels were determined by the DCFH-DH assay. (C) For determination of macrophage paraoxonase 2 (PON2) lactonase activity, cells (2 x 10e) were incubated with 1 mmol/L dihydrocoumarin in Tris buffer, and the hydrolysis rate was determined after 10 min of incubation at 25°C. (D) The extent of Ox-LDL (25 pg of protein/ milliliter, labeled with FITC) uptake by the mice macrophages (1 x 10e) was determined by flow cytometry. Results are expressed as mean S.D. of three different determinations. = p < 0.01 versus placebo.

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