Fig. 4. The agedependent changes in the atherosclerotic lesions and plasma OxLDL levels in apoEKO mice. Male apoEKO mice were mainta ined on a chow diet for up to 40 weeks. A: Whole aortas were stained with oilred O and the percentage lesion area in the aortic sur face was calculated. B: The LDL fraction was separated from each mouse plasma sample. OxLDL levels in the mouse LDL fractions were measured using the antiOxPC mAb DLH3. Data for each mouse (circle) and the mean ± standard deviation (square) are indicated. a, p<0.01 from the value at 4 weeks. b, p<0.05 from the values at 10 and 20 weeks. c, p<0.01 from any other values determined. (Cited from ref. 28 in a modified form with permission by the publisher.) This kit is based on a competitive ELISA using a single anti- modified apoB mAb. It should be noted, however, that their method has quite different features from ours in two respects. First, their mAb binds to a hidden peptide fragment of the apoB protein which is stretched out along the OxLDL surface by a conformational change of apoB.(24) Strictly speaking, this epitope is exposed to the surface of the LDL particles either by oxidation or other modification of apoB. Second, the assay procedure is a competitive ELISA using only one mAb, suggesting that the specificity of the assay may not be sufficiently high to detect very small amounts of OxLDL in the circulation. Some research groups have pointed out that this ELISA kit failed to detect differences between AMI patients and healthy subjects.(25) Behavior of OxLDL during Atherogenesis It has been an ongoing debate for many years whether OxLDL is the cause of atherosclerosis or the result of plaque formation. It is clear from a number of in vitro studies that OxLDL induces inflammatory reactions by activating many types of cells in the vascular wall, including macrophages, smooth muscle cells and endothelial cells, suggesting these reactions are a strong factor in the promotion of atherosclerotic lesion formation. And it is also very likely that inflammation responses increase reactive oxygen species (ROS) production and may thus initiate oxidation reac- tions of PUFA. We thought it is important to study the early events in the vessel walls which take place in the course of atherogenesis to answer this question. It is reported that early lesions in human coronary arteries appear in subjects as young as 20 years old,(26) but it takes decades to develop large and severe lesions, and cardiovascular events start to occur in the decades of the age of 50’s or 60’s. Since it is not easy to study the very early steps of atherogenesis in humans, we examined apoE-knockout (apoE-KO) mice. ApoE- KO mice have been used worldwide as a model animal of sponta- neous hyperlipidemia and atherosclerosis.(27) ApoE-KO mice were maintained on a normal chow for up to 40 weeks. Very small atherosclerotic lesions appeared as early as 10 weeks, and became extensively enlarged after 20 weeks of age. The plasma OxLDL level in the apoE-KO mice increased 4 temporarily at 20 weeks and then decreased to the basal level. This increase in plasma OxLDL level was observed before the expansion of the lesion area (Fig. 4).(28) This observation suggests that LDL is oxidized even in the absence of atherosclerotic lesions, and that OxLDL may be involved in the subsequent development of the lesions. We detected two oxidation epitopes, OxPC and acrolein, in the aortic intima of the apoE-KO mice as early as 4 week of age. One interesting question is why the plasma OxLDL level decreases after the temporal rise at 20 weeks of age. Since the OxLDL appeared to accumulate in the aortic lesions during the expansion of the lesions, it seems highly likely that OxLDL is transferred from the circulation into the vessel wall tissues. Another question is why OxLDL increases in the plasma, since it is thought that LDL is oxidized in the vessel wall tissues and the serum itself has a strong antioxidant capacity. It is possible that OxLDL may easily move between the circulation and vessel wall tissues. Witztum and colleagues demonstrated that the OxLDL levels in the plasma and in lesions change during the progression and regression of aortic lesions in cynomolgus monkeys.(29) Their observations also strongly suggest there is active transport of OxLDL between the circulation and vessel wall tissues. The Presence of OxPC in Pathological Lesions Age-related macular degeneration (AMD) is characterized by a progressive degeneration of the neurosensory retina, retinal pigment epithelium and choriocapillaris in the macular area. The number of patients increasing and AMD has become a leading cause of blindness in the elderly population in Japan. Kamei et al.(30) found a strong positivity to the mAb DLH3 in retinas from patients with AMD. OxPC was much less commonly found in the healthy retina even though the donors were older. The pres- ence of OxPC-modified proteins in the retina was also confirmed by Western blotting. An accumulation of OxPC was found in the chondrocytes in cartilage from patients with osteoarthritis.(31) Osteoarthritis is a local inflammatory disease in joint synovial membranes. Cartilage is a unique tissue in which few cells are scattered in a very thick layer of extracellular matrix and there are very few vessels. doi: 10.3164/jcbn.1100020R1 ©2012 JCBN