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Atherosclerotic lesion formation starts during fetal development and progresses with age after adolescence. However, atherogenesis during the juvenile period has not been studied thoroughly. In this study, we examined the atherogenic susceptibility of juvenile rabbits to cholesterol feeding. Male New Zealand White rabbits aged 8 (younger group) and 12 (older group) weeks were fed a 0.5% cholesterol-containing diet for 8 weeks, and then their aortic atherosclerotic lesion areas were evaluated. Plasma concentrations of total cholesterol, triglycerides, and phospholipids did not differ between the two groups; however, plasma concentrations of high-density lipoprotein cholesterol were 23% lower in the younger than in the older group. Atherosclerotic lesion areas were significantly larger in the younger group (32±21%). However, only moderate changes were observed in these areas in the older group (3.3±0.3%). Histological examination showed marked intimal thickening and macrophage accumulation in the aortic lesions of rabbits in the younger group. To the best of our knowledge, this is the first study to show that dietary cholesterol-induced atherogenic changes markedly occur during a short period in juvenile rabbits.

To study how cholesterol accumulates in atheroma, novel monoclonal antibodies were developed, using crude homogenate of atheroma as immunogens. 212D monoclonal antibody recognizing extra cellular matrix with lipid-laden deposits was selected by histochemical staining. The antigen was deduced vitronectin from cDNA library. DLH3 monoclonal antibody recognizing oxidized LDL, epitope of which was 5- or 9-phosphatidylcholine. Significant correlations between oxidized LDL and coronary heart disease (CHD) patients were observed from clinical study. 256C monoclonal antibody recognizing atheromatous lesions in human aorta was selected. Epitope must be PC-cholesterol complex which may involve in foam cell rupture. Atherogenesis will be discussed from the aspects of these antibodies. Our working hypothesis is required to elucidate the mechanism. Denatured lipoproteins (either oxidized lipoprotein or ruptured foam cells) may induce atheroma. Oxidation of lipoprotein may be taken place both in foam cells and/or extra cellular matrix, and macrophage eliminate these denatured lipoproteins and become foam cells. The foam cells are ruptured by either apoptosis or necrosis afterward, and hydrophobic fragments of foam cells dispersed in extra cellular space, which destroys the function of biological membrane. Since biological function could be maintained by segregation of hydrophilic circumstances, macrophages uptake these fragmented material and oxidized lipoprotein to maintain the function. This vicious spiral may enhance chronically the atheroma.

Oxidized low-density lipoprotein (OxLDL) has previously been thought to promote atherogenesis through foam cell formation. However, the actual nature of OxLDL present in vivo remained obscure until recently. We have produced a monoclonal antibody, DLH3, which specifically binds to OxLDL but not to native LDL. The presence of OxLDL in the LDL fraction of human plasma was demonstrated by introducing a sandwich ELISA procedure using DLH3 together with an anti-apoB antibody. Furthermore, OxLDL levels appeared to increase in certain pathological conditions including acute myocardial infarction and carotid artery atherosclerosis. Accumulation of OxLDL in atherosclerotic lesions has also been demonstrated by immunohistochemical and biochemical studies using the DLH3 antibody. This antibody recognizes oxidized phosphatidylcholines (OxPC) generated during oxidative modification of LDL, and OxPC-apoB adducts formed in OxLDL are the presumed antigens. Measuring OxLDL in plasma would be a useful diagnostic tool for cardiovascular diseases. However, there still remain some major questions related to OxLDL, the answers to which are crucial for understanding the mechanisms of atherogenesis.

Stimulated phospholipid degradation was observed in liver homogenates harvested from rats who had received an injection of carbon tetrachloride (CCl4). When the liver homogenates obtained from CCl4-treated rats were incubated for 1 h at 37°C, approximately half of the phosphatidylethanolamine (PE) was hydrolyzed, producing a stoichiometrical amount of lysophosphatidylethanolamine (LysoPE). The homogenates obtained from control rats showed only poor hydrolytic activity toward endogenous PE. The fatty acid composition of lysoPE produced was essentially identical to that at the sn-1 position of PE, suggesting that A2 type of phospholipase (PLase A2) was involved in the degradation during incubation. The hydrolysis of endogenous PE was dependent on calcium ion. The hydrolytic activity was almost exclusively associated with the membrane fraction which was precipitated by centrifugation at 10000 × g, and did not require the cytosol fraction. Para-bromophenacylbromide, rabbit antibody against rat 14 kDa type II PLase A2, and thielocin A1, a specific inhibitor of type II PLase A2, suppressed the hydrolysis almost completely. These results indicate that the accelerated degradation of endogenous PE observed in liver homogenates of CCl4-treated rat may be mainly due to the activity of the enzyme closely related to type II PLase A2.