Recently, the progression of atherogenesis has been clarified biochemically as well as morphologically. In this study, we would like to investigate the etiology of atherosclerosis utilizing three different models.
(1) Transport of lipoproteins through endothelial monolayer and accumulation of cholesteryl ester in foam cells were examined in vitro.
(2) Monoclonal antibodies recognizing atherosclerotic lesions may help in understanding the progression from both morphological and biochemical aspects.
(3) Transgenic mutant mice may be useful in confirming biochemical evidence with those obtained from other animal models.

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The effects of apolipoprotein E phenotype and dietary P/S ratio on changes in plasma lipoprotein level induced by cholesterol feeding were studied.
1. Apoprotein E phenotype and cholesterol feeding.
Cholesterol (750 mg/day) was fed to healthy volunteers and patients of CVD and CHD for two or three weeks. Apoprotein E phenotype was determined by IEF of VLDL apoproteins. VLDL, IDL, LDL and HDL were fractionated ultracentrifugally and cholesterol levels were measured before and after cholesterol feeding. Changes more than 10% of pre-cholesterol feeding level were considered to be significant, and the trends of LDL-C and HDL-C changes were compared between the subjects with E 3/3 and E 4/3. LDL-C increased 50% in E 3/3 and 33% in E 4/3. We did not find that cholesterol feeding increased plasma LDL more easily in E 4/3 than in E 3/3. Interestingly, cholesterol feeding increased plasma HDL-C levels more frequently in E 3/3 than E 4/3. Increases in HDL-C was found in 40% of E 3/3 and 16% of E 4/3.
2. Dietary P/S ratio and cholesterol feeding.
Patients with CVD and CHD were fed diets of 1, 800 kcal/day (protein 20%, fat 25%, carbohydrate 55%). P/S ratio 0.6, 1.2 and 2.4 of the diet was changed every four weeks. Cholesterol (750 mg/day) was also fed for the latter half of each dietary period. Plasma lipids, apoproteins, cholesterol in lipoproteins and activities of LPL and HTGL in the postheparin plasma were measured at every two weeks. Plasma LDL-C increased on the diet of P/S 0.6. The changes of LDL-C were variable on the diet of P/S 1.2 and 2.4. The changes of LDL-C mainly depended on the light LDL (LDL₁), HDL-C did not decrease even on the diet of P/S 2.4. Cholesterol feeding did not alter plasma total HDL-C levels but HDL₂-C/HDL₃-C ratio increased on the diet of P/S 0.6 by cholesterol feeding. Cholesterol feeding stimulated the activities of LPL on the diet of three different P/S ratio. It also stimulated HTGL activities on the diet of P/S 1.2 and 2.4 but not on the diet of P/S 0.6. HTGL activity/LPL activity ratio had a negative correlation with LDL₁-C/LDL₂-C ratio (r=-0.348, p<0.05) and HDL₂-C/HDL₃-C ratio (r=-0.467, p<0.01). HTGL and LPL must be important for LDL distribution as well as for HDL distribution.

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Regular physical exercise was associated with significant increases in HDL-cholesterol levels and lower LDL-cholesterol levels. Endurance training such as jogging increased HDL-cholesterol significantly. Smoking was associated with lower HDL-cholesterol and daily moderate intake of alcohol was associated with higher HDL-cholesterol. In subjects who showed an increase in plasma triglyceride and delayed removal of chylomicrons and chylomicron-remnants after the consumption of alcohol (0.4 g/kg/day) for 4 weeks, HDL-cholesterol did not increase. The ability to remove triglyceride rich lipoproteins seemed to influence the response to alcohol loading.
HDL-cholesterol and plasma AI and AII increased significantly 3 weeks after the consumption of filtered coffee (5 cups/day) in healthy subjects. The coffee with low content of caffeine showed a similar effect in raising HDL-cholesterol. This suggests that components other than caffeine are responsible for the changes.
Data concerning the effects of commonly prescribed antihypertensive drugs on blood lipids and apoproteins were reported.
Regular physical exercise, smoking, alcohol, coffee drinking, and the administration of some antihypertensive drugs have a considerable influence in the development of atherosclerotic diseases by modulating ulasma livid metabolism.

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We summarized the effects of drug therapy on hyperlipidemic patients, based on our clinical experiences and reviews of several international literatures.
In general, the following facts are known:
(1) Incidence of CHD can be reduced by lowering cholesterol, especially LDL-C.
(2) First, we must exclude secondary hyperlipidemia. Then, we should begin with diet therapy and, if it is not so effective, follow with antihyperlipidemic drugs.
(3) Incidence of CHD appears to be related more with an increase in LDL-C than a decrease in HDL-C.
(4) Some atherosclerotic lesi ons regress by therapy.
The following problems must be solved, particularly in Japan.
(1) Real efficacy of antihyperlipidemic drugs on atherosclerosis in Japanese must be investigated by long double-blind, controlled trials.
(2) The level of cholesterol not only to prevent atherosclerosis but also to induce regression of atherosclerotic lesions must be established in Japanese.
(3) It is important to develop effective and safe reatment (s) for intractable cases of various drug therapies.

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It has been established previously that low density lipoprotein (LDL) incubated with cultured endothelial cells, smooth muscle cells, or macrophages undergo a free radical-catalyzed oxidative modification. This modification involves lipid peroxides and extensive structural changes in the LDL molecule. The oxidatively modified LDL inhibits the motility of the mouse resident peritoneal macrophages, yet lysophosphatidylcholine in oxidized LDL acts as a chemotactic factor for circulating human monocytes. The present study demonstrated that the stimulation of human mononuclear cells with phorbol myristate acetate (PMA) results in an increase of oxidized LDL, which was assayed by LPO-KIT (obtained from Kyowa Hakko Inc.) in the culture medium. The medium containing oxidized LDL did not exhibit a chemotactic for human monocytes, but inhibited the chemotactic responses of monocytes to the N-formyl peptide and aortic extract.
These findings suggest that circulating monocytes may play a role in the subendothelial space by some chemotactic factors and become trapped in the splace due to the inhibitory effects of oxidatively modified LDL.

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We investigated the effect of Probucol treatment on regression of xanthelasma. The subjects were classified into three groups: a group of familial hypercholesterolemia (FHC), non FHC-hyper-cholesterolemia (non FHC-hyper TC) (TC≥230 mg/dl), and non FHC-normocholesterolemia (non FHC-normo TC) (TC<230 mg/dl), based on the plasma cholesterol levels and the etiology of the disease. In the FHC group, the serum total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), and apolipoprotein B (apo B) levels were significantly higher; and the serum high density lipoprotein-cholesterol (HDL-C) and apo A-I levels were significantly lower in the subjects with xanthelasma than in those without xanthelasma. In addition, the frequency of ischemic heart disease was higher and the Achilles tendon was thicker in the former than in the latter. In the non FHC-hyper TC group, the serum apo B level was significantly higher and the apo A-I level was lower in the subjects with xanthelasma than in those without xanthelasma. In a group of non FHC-normo TC, the serum HDL-C, apo A-I and apo E levels were significantly higher in subjects with xanthelasma than in those without xanthelasma. In all groups, xanthelasma were tend to regress after 3 months of Probucol treatment and the regression or disappearance of xanthelasma was observed in a few cases after 12 months.

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(1) Guanfacine was administered 1.0 mg/day to 11 patients with essential hypertension resulting in: 1) both systolic and diastolic blood pressure decreasing significantly after 4 and 12 weeks of treatment, 2) pulse rate and plasma concentrations of β-TG and PF 4 did not change significantly, 3) platelet aggregation was unchanged following administration of 5.0, μM of ADP, 2.0, μg/ml of collagen, or 2 mM of arachidonic acid, but it tended to decrease with 9.1, μM of epinephrine after 12 weeks of the treatment.
(2) In vitro study using healthy volunteers showed that 1) guanfacine dose-dependently inhibited epinephrine-induced platelet aggregation, 2) guanfacine dose-dependently potentiated platelet aggregation induced by low concentrations of ADP, and this effect was inhibited by yohimbine but not by prazosin.
From these results, it was suggested that the action of guanfacine was mediated by α₂-adreno-ceptor of platelets.

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The activity of platelet-activating factor (PAF) acetylhydrolase in plasma was estimated in 18 healthy male habitual cigarette smokers and 21 healthy age-matched nonsmokers. A special reference was made with the activity with serum lipoproteins (LDL and HDL) that had been known to carry the enzyme. The average value of plasma PAF acetylhydrolase activity in smokers was 63±52.9 nmol/ml/min, which was significantly higher than that in controls: 34±26.3 nmol/ml/min. There were no significant differences in serum lipids or lipoproteins between the two groups, except the ratio of apoprotein B to apoprotein A-I (Apo B/Apo A-I), which was higher in smokers. In smokers, plasma PAF acetylhydrolase activity correlated positively with serum total cholesterol, LDL-cholesterol, triglyceride, apoprotein B, LDL-cholesterol/HDL-cholesterol ratio, and Apo B/Apo A-I; and negatively with serum HDL-cholesterol and apoprotein A-I. None of these correlations was significant in nonsmokers. In addition to the increased activity, the relative distribution of the enzyme activity among lipoprotein classes may be altered in habitual smokers. Such alterations were already evident in healthy smokers without any clinical manifestation of atherosclerotic diseases. PAF may play a role in smoking-induced vascular injuries.

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Microfocusing postmortem coronary angiography was used to detect early atherosclerotic lesions in young people with no known risk factors. Following isolation and saline washing, coronary artery specimens were placed on a special radiolucent board, which could be rotated along the long axis so that mild eccentric stenosis could be identified. Angiography was performed with microfocusing image using radiopaque Barium. Ring sections were obtained at stenotic sites and compared with the angiographic findings. Using this method, we were able to see trivial eccentric stenosis not normally observed during macrographic postmortem angiography or premortem coronary angiograms.
One hundred twenty-one autopsy cases were studied. Cases with evidence of cardiovascular disease (chest pain, palpitation, hypertension, EKG abnormalities, cardiomegaly on chest X-Ray, etc.), diabetes mellitus, or hyperlipidemia were eliminated from this study.
The cases were divided into two groups: 68 cases (Group A) were examined macro-and microscopically after the coronary arteries were cut open and fixed. 53 cases (Group B) were examined by the microfocusing method without opening the lumen of the coronary arteries.
In group A, 27 cases (39.7%) had at least one significant atherosclerotic lesion. In group B, 42 cases (79.2%) had visible stenosis by angiography. In group B, 23 cases (43.4%) showed more than 50% stenosis, while 19 cases (35.8%) had lesions with less than 50% stenosis, including very mild eccentric stenosis that might not be detected by macrographic methods. In both groups the left anterior descending artery (LAD) had the highest incidence of stenosis and most of the cases with two or three vessel disease had at least one LAD lesion.
The degree of stenosis on angiography was compatible with findings on ring sections. Histologically, severe stenosis was mostly associated with duplication of the internal elastic lamina, foam cell formation, and core necrosis. On the other hand, mild eccentric stenosis was mainly associated with focal intimal thickening without changes in the internal elastic lamina or foam cell formation. Cardiac weight, interventricular septum thickness, anterior and posterior ventricular wall thickness, and degree of obesity showed no correlation with incidence and severity of coronary atherosclerosis.
Stratifying by race and sex, black males had significantly higher incidence and severity of coronary atherosclerosis than other groups. No positive correlation could be seen between severity of coronary stenosis and aortic atherosclerosis.

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Very low density lipoprotein (VLDL)-triglyceride (TG) kinetic studies were performed using puromycin-induced nephrotic rats to investigate the mechanism of hypertriglyceridemia in nephrotic syndrome. Nephrotic rats recorded 6 times higher plasma and VLDL-TG concentrations than the controls. VLDL-TG was endogenously radiolabeled in the donor rats with [2-³H]-glycerol. The half life (T 1/2) was then determined by monitoring the clearance of plasma radioactivity of TG in the recipient rats. When [³H]-VLDL-TG from the controls was re-injected into the control recipients, [³H]-VLDL-TG rapidly disappeared from the plasma (T 1/2=3.5min) in the first order kinetics. In contrast, when [³H]-VLDL-TG from the nephrotic rats was re-injected into the nephrotic recipients, [³H]-VLDL-TG was not eliminated efficiently and 96% of the tracer still remained 10 min after injection. When [³H]-VLDL-TG from the nephrotic donor was re-injected into the control recipients, T 1/2 of [³H]-VLDL-TG was significantly longer (6.7min) than that of the control [³H]-VLDL-TG (p<0.02). The TG secretion rate in the nephrotic rats determined by the Triton WR-1339 technique was only 1.2 fold higher than controls. Lipoprotein lipase activities were examined in the post-heparin plasma of the control and the nephrotic rats. There were no significant differences in the activities between the two groups.
We suggest that in addition to an increase of hepatic production of TG, the abnormal VLDL particles, due to nephrotis, results in the catabolic defect of TG which leads to substantial hypertriglyceridemia.

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γ-Oryzanol has been widely used for hyperlipidemia and lowers the serum cholesterol. It has been reported that γ-Oryzanol improves cholesteryl ester metabolism in the arterial wall. The aim of this study is to investigate the effects or γ-Oryzanol on the atherosclerotic lesions. The carotid arteries of Wistar rats were de-endotherialized with a balloon catheter, the rats were then divided into 4 groups (fed with normal diet (ND), atherogenic diet (AD): 2% Cholesterol, 0.5% Cholic-Na, 0.2% Propylthiouracil, 10% Lard, 5% Sugar, and AD containing 0.5%, and 1.0% γ-Oryzanol for 13 days). The atherosclelosis induced in AD group rat resembles human atherosclelosis (i.e. intense intimal thickening with cholesteryl ester accumulation and appearance of lipid laded form cells).
The total cholesterol and cholesteryl ester in the artery of the 4 groups are shown below. (Table)
γ-Oryzanol significantly inhibited the cholesterol accumulation in the atherosclerotic lesions. Analysis of the serum lipid showed a decrease in total, VLDL-and LDL-cholesterol, while there was no change in HDL-cholesterol. We also investigated the effect of γ-Oryzanol on intimal thickening induced by carotid artery de-endotherialization, γ-Oryzanol tended to inhibit intimal thickening. From these results, we suggest that γ-Oryzanol suppresses atherosclerosis.

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