There is a great deal of evidences that cholesteryl ester accumulates in the intra- and extracellular space in atherosclerotic lesions. In this paper, we have proposed that cholesteryl ester accumulated in the lysosome in an unhydrolyzed form and transferred from the lysosome to the cytoplasm, in addition to the re-esterification of cholesterol in the microsome. We also proposed that a part of the cholesteryl ester deposited in the extracellular space may be derived from ruptured highly lipid-filled foam cells.

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Interrelations between arteriosclerosis and spasm will be discussed, with special reference to the coronary arteries. Coronary spasm might be experimentally induced by a 50% reduction in blood flow by coronary ligation in dogs. This might leads to the idea that arterial stenosis might play a role in the pathogenesis of coronary spasm. It has been known that drugs or biological activators such as ergonovine, histamine, serotonin, etc. can induce arterial spasm. Although cholinergic agents allow other arteries to dilate in general, it has been confirmed that methacholine, a cholinergic agent, can let coronary arteries contract or even be spastic at least in the dog and pig.
We produced coronary sclerosis within 3 months after rubbing on the intimal surface of the anterior descending branch of the coronary arteries. In such sclerotic arteries, spasm could be easily induced by methacholine injections in a significantly higher frequency than in the non-rubbed, nonsclerotic control. However, spasm occurrence was not related to the time period after intimal rubbing or to the severity of coronary sclerosis, During our later experiments, it has become clear that spasm can occur immediately after intimal rubbing and subsequent methacholine injections, and is unrelated to intimal thickness. This may indicate that endothelial denuding or intimal injury may play a certain role in the pathogenesis of coronary spasm following loss of endothelium-derived relaxation factor (EDRF, Furchgott) and/or platelet aggregation, rather than blood flow reduction caused by intimal thickening.
In a trial to induce frequent and repeated coronary spasm by multiple methacholine injections in swine, vascular permeability became accelerated in the spastic segments of the arteries, as shown by Evans blue infiltration, anti-fibrinogen and antialbumin immunostains, intimal thickening with edema, and ferritin-tracing electron-microscopy.
Thus, coronary spasm and sclerosis might be considered to influence each other and to constitute a vicious cycle.

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There are many reports about the effects of Ca-channel-blockers or Ca-chelating agents on experimental atherosclerosis. These results have not always indicated positive effects on inhibition of atherogenesis. Although some explanations about the above-mentioned complicated results have been proposed, there are many problems remaining to be solved. For example, the differences may be due to differences in the doses, administration route or the mechanisms of activities of the Ca-antagonists. It is known that some Ca-antagonists exhibit inhibitory activities on platelet aggregation even at ordinary doses. If we can use more potent Ca-antagonists, we might obtain uniform results regarding their antiatherogenic activities. Furthermore, it is expected that the results of primary or secondary prophylactic trials by Ca-antagonists for coronary heart disease, which are now under way, may lead to a conclusion.

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The vascular endothelium is known to act as a selective barrier to macromolecules in the blood. To clarify the molecular mechanism of transport through the endothelial cell monolayer, we developed an in vitro model system, consisting of porcine arterial endothelial cells cultured on type I collagen gel supported by a dacron sheet. In this study, the mechanisms of transport of lipoprotein labeled with rhodamine B (RB-LDL) and fluorescein dextran (FD; 4K, 10K, 20K, 70K, 150K) through the endothelial cell monolayer were studied using this system. RB-LDL transport at 37°C was dose-dependent saturable at 0.4mg protein/ml, but little occurred at 0°C. Concerning the transport of FD, the rate of transport through the monolayer depended on the size of the FD. FD transport was not temperature-dependent and was not a saturable process.

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In previous studies, we have reported that free and crystalline cholesterol activated human complement through the alternative pathway. Furthermore, rabbit aortic smooth muscle cells (SMC) seemed to activate human complement through the alternative pathway and bind with human C3 and IgG in the immunofluorescence technique.
In the present study, the following were examined.
1) C6-9 complex was extracted into phosphate buffered saline from human atheromatous tissue and identified by enzyme linked immunosorbent assay (ELISA). This complex was the same as the C5b-9, which is the so-called membrane attack complex (MAC).
2) Free and crystalline cholesterol activated the human complement system and formed the C6-9 complex in the consequently conducted ELISA.
3) Human atheromatous tissue more strongly converted human C3 through the alter native pathway than did human normal aortic tissue.
4) Swine SMC exhibited a tendency to activate the human complement through the alternative pathway and bind with human C3, the same as in the case with rabbit SMC.
5) Intact cultured SMC obtained from rabbit and swine were not injured by activation of the human complement. The reason is considered to be that MAC did not form at the surface of the SMC, since the inhibitor of complement activity may exist in situ.
These results suggest that the complement system may be inplicated in the initiation and progression of atherosclerosis. However, a further study should be conducted to allow valid conclusion to be drawn.

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The adherence of monocytes to the endothelium of large arteries and their subsequent migration to the intima, followed by modulation into lipidladen macrophages are early events in diet-induced atherogenesis. In this study we have examined the interaction of rat peritoneal macrophages with cultured vascular endothelial cells, as well as macrophage-derived chemotactic activity of macrophages. It was found that adhesion of macrophages to endothelial cells was dependent on the presence of divalent cations, and was inhibited at 4°C. Pretreatment of endothelial cells with LDL and VLDL significantly increased subsequent adhesion of macrophages to these endothelial cells. In addition, macrophages from hypercholesterolemic animals showed enhanced attachment to the endothelial cells; they also moved more quickly when examined by time-lapse cinephotomicrography. Macrophages in culture secreted potent chemotactic factors for macrophages. This production of chemotactic factors by macrophages and chemotactic properties of macrophages to the autoconditioned media were remarkably increased when animals were exposed to hypercholesterolemia. The results suggest that in hypercholesterolemia, macrophages are more adhesive to endothelial cells and tend to accumulate in the intima. It seems likely that macrophagederived chemotactic activity for macrophages leads to the recruitment and retention of monocytes/macrophages in the arterial wall.

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To elucidate the role of HDL in cholesterol metabolism of macrophages or macrophagederived cells, HDL interaction with rat peritoneal macrophages and sinusoidal liver cells was studied. Specific questions addressed in the present study were two-fold; functional correlation of HDL receptor with HDL-mediated cholesterol transfer to macrophages, and post-binding fates of HDL.
HDL receptor and its functional correlation with HDL-mediated cholesterol transfer
Binding at 0°C of ¹²⁵I-HDL to these cells demonstrated the presence of a saturable membrane-associated receptor. Upon chemical modification of tyrosine residues of HDL apoprotein by tetranitromethane (TNM), the specific binding was abolished. However, further reduction of TNM-HDL with persulfite led to a significant recovery of the ligand activity. Cholesterol transfer from HDL to these cells was reduced by 50% by TNM-treatment. These findings indicate the possible involvement of specific binding in HDL-dependent cholesterol transfer to the cells.
Receptor-mediated endocytosis via a nonlysosomal pathway
HDL incubation with these cells did not result in intracellular degradation of HDL apoprotein. Upon acetylation, however, HDL underwent lysosomal degradation. Upon chase experiments, the cell-associated HDL obtained after incubation at 37°C was released into the external medium as an acid-precipitable form. Moreover, the measurement of microenvironment pH of the ligand by FITC-labeled HDL disclosed that the ligand was located in a much more acidic compartment upon 37°C-incubation than 0°C-incubation, and this acidic pH was dissipated by CCCP, a well-known protonophore. These results suggest, therefore, that the cell surface-bound HDL particles were internalized into the cells and exposed to an acidic environment, and were delivered back into the external medium, rather than routed to the lysosomes for degradation. This notion was further supported by a morphological approach using HDL conjugated with horse radish peroxidase.

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In order to observe the effects of milk administration on serum lipoprotein levels in newborns, serum lipids and apolipoproteins (apo A-I, A-II, A-IV, B, C-II, C-III and E) were determined successively in the early neonatal period. Levels of apo A-I, A-IV, B, C-II and C-III significantly increased during the period compared to the initial levels at birth. In particular, apo A-IV level significantly increased from 6.9±1.0mg/dl on day 0 to 8.1±1.6 (p<0.05) and 15.7±2.8mg/dl (p<0.001) on day 3 and 6, respectively. Serum apo A-I also increased during the period, though it did not change markedly compared to apo A-IV. Although apo B level significantly increased during the period, it is unclear which of the apo B species, apo B-48 or B-100, is more responsible for the increase in the present study. Conversely, apo A-II and E levels did not change significantly during the period. Apo A-IV increased most markedly during the period compared to those of other apolipoproteins determined in this study, suggesting that the production of apo A-IV in the intestine was increased considerably during fat absorption by the lactation.

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Thirty-one year-old male had been complainting of nocturnal dyspnea for one week before admission to our hospital. He smoked thirty cigarettes a day for 11 years and consumed no alcohol. His father died from myocardial infarction at the age of 56, and two of his father's sisters died of heart disease. One of his two children has VSD.
Physical examinations on admission revealed that the pulse rate was 100beats/min. and regular, and that the blood pressure was 132/88mmHg. Moist rales and galloping rhythms were heard. No murmurs were heard. The liver and spleen were not palpable. No peripheral edema, nor cyanosis were noted, and no xanthom was noticed.
The cholesterol and triglyceride levels in serum were 380mg/dl and 149mg/dl, respectively. Abnormal thickness of the Achilles tendons was also demonstrated on an X-ray image. An X-ray image of the chest revealed a butterfly shadow associated with massive consolidation at the right lower lobes. On an electrocardiogram, abnormal q wave in leads I, II, aV_L, V_4-6 and inverted T wave in leads I, aV_L, V₅ and V₆ were seen.
An echocardiogram showed extensive hypokinesis at the LV wall, LV dilatation and 0.39 of ejection fraction. A ²⁰¹Tl scintigram revealed perfusion defects in the apex and lateral wall. The findings of a coronary angiogram showed that there was 90% stenosis in segment 6, total occlusion in segment 7, 99% stenosis in segment 12, and coronary aneurysm in segments 2 and 3. There was good development of collateral flow from the right coronary artery to the left anterior descending coronary artery. The left ventriculogram revealed akinesis in segments 2, 3, 4 and 7, and hypokinesis in segments 1, 5 and 6. He was diagnosed to have familial hypercholesterolemia associated with coronary aneurysm and subsequently to have myocardial infarction. He is now well and the cholesterol level in his serum is being controlled well by LDL apheresis (1/2 weeks) and Probucol (500mg/day, b. i. d.).
It is suggested from this case that one should consider the possibility that myocardial infarction in juveniles may be complicated by familial hypercholesterolemia.

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This study was performed to analyze the relation between coronary risk factor and nutrition. The subjects included 663 apparently healthy automobile factory employees. Items measured in this study included serum lipids, resting blood pressure and resting ECG. Nutritional values and smoking were investigated by questionnaires. The results showed an inverse correlation between HDL-cholesterol and dietary carbohydrates, triglyceride and uric acid increased with alcohol intake and a strong correlation between obesity and coronary risk factor was noted.
From the results of a quantification method applied to multi-dimensional qualitative data, the major factors of the first and second components were found to be blood pressure and serum cholesterol, respectively. Blood pressure and serum lipids were shown to be independent factors in relation to ischemic changes on electrocardiograms.

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It is important to evaluate the metabolism of exogenous lipoproteins because chylomicron remnants have been recognized to be atherogenic. We prepared three kinds of test meal as follows: BI; carbohydrate (C) 63g, protein (P) 18g, long chain triglyceride (LCT) 4.3g. BII; C 53g, P 18g, medium chain triglyceride (MCT) 8g, LCT 5.5g. Bill; C 53g, P 18g, LCT 13.5g. We administered these test meals to four healthy men (mean age 30 yrs.) and obtained their plasma after 0, 1, 2 and 3 hours of loading.
After administration of BI and BII, the apolipoprotein B-48/B-100 ratio in triglyceride rich lipoproteins (TRL, d<1.006), which was determined by analytical SDS polyacrylamide gel electrophoresis, increased to the maximum value after one hour and thereafter rapidly decreased. In contrast, on loading of BIII, this ratio consistently increased during the course of the experiment. Plasma insulin levels behaved like the apo B-48/B-100 ratio: large insulin responses were found after administering BI and BII while the obvious peak of insulin responses was not observed after feeding BIII.
Although the apo B-48/B-100 ratio in TRL decreased after reaching its maximum value, triglyceride levels in TRL continued to increase throughout the period of experiment on BI and BII, reflecting stimulated secretion of endogenous very low density lipoprotein. On the other hand, total cholesterol levels in low density lipoprotein (LDL) decreased after administration of BI and BII probably due to increased activity of LDL receptors during the postprandial phase.
It is worth nothing that the clearance of intestine originated lipoproteins was greatly affected by differences in food composition, especially free fatty acids. It is likely that this metabolic process was mediated through insulin responses.

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The effects of simvastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, were investigated in cholesterol-fed rabbits.
Experimental rabbits were divided into 4 groups. Group A received simvastatin (10mg/kg/day) orally for 5 weeks and group B received the same dose for 10 weeks (simvastatin groups). The control groups C and D received a placebo for 5 and 10 weeks, respectively. The levels of serum total cholesterol (T-C), triglyceride (T-G), β-lipoprotein and high density lipoprotein cholesterol (HDL-C) were measured every week. At the end of the experimental period the aortas were removed, opened longitudinally and stained with Sudan III.
Both placebo groups showed a significant increase in T-C, T-G and β-lipoprotein concentrations, but the simvastatin groups did not show such an increase. There were no significant differences in HDL-C levels between the simvastatin and placebo groups. Integrated values of T-C (Integrated T-C, mg·day/d/) were 8, 318.1 in group A, 10, 152.1 in group B, 28, 754.6 in group C and 73, 430.0 in group D. Aortic surface involvement (S·I, %) was 0.5 in group A, 0.3 in group B, 7.5 in group C and 16.6 in group D. S·I was related closely with the integrated T-C (r=0.899).
These results demonstrate the suppressive effects of simvastatin on atherogenesis by reducing hypercholesterolemia.
Total lipids in feces excreted during a 24-hour period were measured. Fecal lipids had increased two-fold in the simvastatin group (5mg/kg/day). It was suggested that simvastatin may accelerate the lipid excretion into the intestine.

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Studies were designed to explore the relationship between functional abnormalities of lipoprotein lipase (LpL) and type V hyperlipoproteinemia in two cases. Patient A was a 34-year-old woman with a plasma triglyceride (TG) level of 6, 200mg/dl during pregnancy and who suffered from acute pancreatitis. Patient B was a 14-year-old girl with a plasma triglyceride (TG) level of 2, 200mg/dl and who also suffered from acute pancreatitis. LpL was purified from Postheparin plasma using heparin-Sepharose and phenyl-Sepharose. The LpL from both patients had hydrolyzing activities toward the water soluble substrate, tributyrin.
The hydrolyzing activities toward Triton X-100 emulsified triolein and chylomicron-triolein by 1U Esterase of normal LpL were 97.5n moles/h and 39.0n moles/h, respectively, whereas those by 1U Esterase of patient A's LpL were 95.5n moles/h and 14.5n moles/h, respectively. And 1U Esterase of patient B's LpL had almost no hydrolyzing activities toward Triton X-100 emulsified triolein and chylomicron-triolein. These results suggest that patient A has abnormal LpL which cannot recognize chylomicron, and that patient B has abnormal LpL which cannot recognize long chain fatty acid esters.

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The morphological changes of coronary arteries in cases of myocardial infarction were analyzed by morphometric techniques and a tensile test. Morphometrically arterial stenosis, which was quantified by the stenotic index [(1-luminal area/total arterial area)×100], did not increase linearly with the enlargement of the intimal area. The stressstrain curves measured in the left coronary arteries indicated an increase of arterial distensibility in parallel with increasing strain. This arterial distensibility may be morphologically correlated with collagen and elastic fiber disorientation in the thickened intima. This increased arterial distensibility may be a specific change in the sclerotic coronary arteries of myocardial infarction group compared with the non-myocardial infarction group.

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LDL-receptor activity was examined by the PHA-stimulated proliferation of lymphocytes. When freshly isolated human peripheral blood lymphocytes was incubated in RPMI 1640 containing lipoprotein-deficient serum, PHA-induced ³H-thymidine incorporation into lymphocytes was reduced by 86% in the presence of 0.1mM CS-514, a specific inhibitor of 3-hydroxy 3-methylglutaryl-coenzyme A reductase. The inhibition was overcome by mevalonic acid or a low concentration (1-2μg/ml) of LDL-cholesterol supplementation. However, even a high concentration (10μg/ml) of HDL₃-cholesterol could restore little of the suppressed lymphocyte response. When endogenous cholesterol synthesis was blocked and exogenous cholesterol was provided as LDL, lymphocyte proliferation was dependent on functional LDL-receptors.
We proposed two indices for LDL-receptor activity: the ratio of incorpolated ³H-thymidine to added LDL-cholesterol, especially 1.0μg/ml of LDL-cholesterol, and integrated ³H-thymidine incorporation by LDL-cholesterol administration at doses from 0 to 1.0μg/ml, namely, the area below the dose-response curves of ³H-thymidine incorporation at these doses of LDL-cholesterol. Analyses with these indices showed that the lymphocytes from FH heterozygotes with Achilles tendon xanthoma had a significantly lower level of LDL-receptor activity than those from FH heterozygotes without Achilles tendon xanthoma, non-familial hypercholesterolemic patients and normolipidemic subjects, and that LDL-receptor activity of the lymphocytes from FH homozygot was much lower than that of FH heterozygotes with Achilles tendon xanthoma. These results demonstrate that estimating LDL-receptor activity from PHA-stimulated lymphocyte proliferation is useful for detecting FH homozygot and heterozygotes with Achilles tendon xanthoma, especially when LDL-receptor activity is expressed by the two indices.

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The possible role of plasma insulin levels as a risk factor in coronary artery disease (CAD) has been studied in 51 normolipidemic (total cholesterol<230mg/dl, triglyceride<150mg/dl) male patients aged 34-80 years. Selective coronary angiography (CAG) was performed in all patients on average within one month of the 75g oral glucose tolerance test (OGTT). CAD was defined as a 75% or greater luminal diameter narrowing of one or more of the major coronary arteries. According to the results of CAG, the patients were divided into two groups: patients with CAD (n=31) and normal coronary arteries (NCA: n=20). The coronary risk factors such as age, blood pressure, smoking, and body mass index were matched for the two groups. Plasma glucose and insulin levels before and 30, 60 and 120 minutes after glucose ingestion were determined in the two groups. Plasma insulin response to glucose ingestion was evaluated by calculating the sum of the insulin levels 0, 30, 60 and 120 minutes after glucose loading. The early insulin response to glucose ingestion (ΔIRI) was calculated as the difference in the values between 0 and 30 minutes. The prevalences of diabetes mellitus (DM) and impaired glucose tolerance (IGT) in the NCA group were 15 and 60%, and those in the CAD group were 10 and 71%, respectively. Thus, the prevalences of abnormal glucose tolerance in the two groups were not significantly different. The values of total cholesterol and triglyceride in the CAD group were similar to those of the NCA group. However, a statistically lower HDL-cholesterol value was observed in the CAD group than in the NCA group. The glucose areas (ΣBG), the insulin areas (ΣIRI), basal insulin levels, and the difference in glucose values between 0 and 30 minutes (ΔBG) were not significantly different. ΔIRI and ΔIRI/ΔBG were significantly higher in the CAD group than in the NCA group. The present data support the hypothesis of the direct atherogenic action of high plasma insulin response during OGTT. In particular, a high early insulin response might be a marker of enhanced liability to the evolution of coronary atherosclerosis.

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The effects of dietary cholesterol on plasma lipids were studied in noninsulin-dependent diabetic rats induced by a neonatal injection of streptozotocin. The mild diabetic state was induced by an intraperitoneal injection of streptozotocin (STZ: 90mg/kg body weight) dissolved in 0.05M citrate buffer, pH 4.5. 2 days after birth. A cholesterol-rich diet (1.0%) was started 8 weeks after STZ injection and continued for 4 weeks. The plasma glucose concentration during fasting (183.5±38.4vs. 156.8±17.9mg/dl, p<0.05) and 30, 60 and 120 minutes after glucose ingestion (1.5g/kg body weight) were significantly increased in the diabetic rats compared with the control rats. There were no significant differences in fasting plasma immuno-reactive insulin levels and body weight. Increased intake of the diet was not observed in the diabetic rats. Responses of plasma cholesterol to the cholesterolrich diet were augmented in the diabetic rats compared with the control rats (184.5±11.2vs. 98.4±23.0mg/dl, p<0.05). No significant differences were found in the plasma concentration of triglyceride and phospholipid between diabetic and control rats. The data presented indicated that responses of plasma cholesterol to the cholesterolrich diet were augmented in the nonobese and noninsulin-dependent diabetic (NIDDM) rats.

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