Serum levels of lipoprotein-cholesterol and apolipoprotein were studied in 77 male (57±8yrs.) and 26 female (59±8yrs.) patients who undergoing coronary angiographycally examination for suspected myocardial ischemia. The extent and severity of coronary artery disease (CAD) were assessed by assigning scores to each lesion.
The coronary scores were correlated with the apo B and apo A-I/B ratio in female patients and apo A-I/B ratio in male patients. While, apo A-I, A-II, C-II, C-III, E showed no significant correlation with the coronary scores.
The male patients were divided into four groups based on the coronary scores; H-CAD (range: 11- points), M-CAD (5-10 points), L-CAD (1-4 points), N-CAD (0 point). The atherogenic risk factors other than the abnormalities in lipid metabolism (obesity index, fasting plasma glucose, smoking and blood pressure) were well matched in the H-CAD, L-CAD and N-CAD groups. TC, LDL-C, HDL-C, HDL₂-C, apo B, apo A-I/B ratio, and apo A-II/B ratio were significantly differed in the H-CAD and N-CAD groups. While apo A-I, A-II, C-II, C-III, E were not significantly changed in four groups. Furthermore, apo B and apo A-I/B ratio were significantly differed in H-CAD and L-CAD groups.
These results suggest that apo B and apo A-I/B ratio are useful discriminators of the severity of the coronary artery disease. And lipoprotein-cholesterols appears to be more sensitive than apolipoproteins to reflect the severity of the coronary artery disease.

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One step enzyme immunoassay for apo B has been developed and its clinical significance has been studied. Diabetic, hyperlipidemic, and those with cerebrovascular or cardiovascular disease have been investigated. Serum levels of apo B increased in diabetic subjects even in normolipidemic subjects, but were normalized in well controlled normolipidemic diabetic subjects. Apo A-I and A-II did not change while HDL-cholesterol decreased.
In familial hypercholesterolemia, serum apo B increased 2-3 folds, while in hypobetalipoproteinemia, serum apo B was detected only by EIA and not by SRID method.
In cerebrovascular disease, serum apo B increased with apo A-I being lowered. LDL became apo B rich, since chol/apo B in LDL decreased significantly. Subjects with thrombosis tends to have higher levels of cholesterol and apo B over those with bleeding or embolism. Hyperlipidemia and qualitative abnormality of LDL may affect larger blood vessels more greately than the smaller arteries which seems to be responsible for bleeding or embolism.
In subjects with angina or myocardial infarction, serum levels of apo B also increased. No significant changes of apo A-I have been observed.
By one-step ETA, serum levels of apo B can be determined more accurately and easily (50-60 samples within 4-5 hours). Apo B/apo A-I significantly increased in diabetes, familiar hyperlipidemia (IIa, IIb, IV) and subjects with cerebroor cardio-vascular disease. Apo B rich LDL has been observed in subjects with atherosclerotic cerebrovascular disease.

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Since apolipoprotein E (apo E) has a significant role in lipoprotein metabolism, we investigated apo E phenotype involving plasma lipid levels, and the relationship between apo E and coronary artery stenosis. In addition, clinical features of type III hyperlipoproteinemia discovered recently were studied. For the six phenotype apo E 3/3, E 4/3, E 4/4, E 4/2, E 3/2 and E 2/2, the percentage distribution in 227 healthy appearing subjects were 74.4, 16.3, 1.8, 0.4, 7.0 and 0, respectively. As a result, the frequency of parent form (apo E 3/3) tended to be higher than that in western industrial countries. The incidence of hypercholesterolemics in each apo E phenotype revealed that the percentage of apo E 3/3, apo E 4/3+E 4/4 and apo E 3/2 were 6.5, 9.7 and 18.8, respectively. The number of hypercholesterolemics of the E 3/2 group was significantly higher than that of the E 3/3 group. On the basis of coronary angiography results, the patients were divided into two groups, coronary artery disease (CAD) (+) and CAD (-). No significant difference in the distribution of each apo E phenotype was found. Clinical findings, plasma lipids, lipoproteins and apolipoproteins in five cases with type III hyperlipoproteinemia associated with apo E 2/2 were investigated. The type III hyperlipoproteinemia seems to manifest itself when, in addition to the subjects bearing apo E 2/2, there is either environmental or genetic stimulation.

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The serum levels of apolipoprotein A-IV (apo A-IV) were measured by rocket immuno-electrophoresis in disease-free humans, at fasting and after oral and intravenous fat administration. The study was extended to patients with chronic pancreatitis, malabsorption syndrome, to post-operative patients on total parenteral nutrition and to patients with liver diseases, cholestasis, diabetes mellitus and chronic renal failure. Oral fat ingestion resulted in an increase of apo A-IV levels which remained elevated even when the postprandial hypertriglyceridemia had disappeared. A transient increase in apo A-IV levels was observed after intravenous fat infusion but the level declined simultaneously with decreases in triglyceride levels. Levels of serum apo A-IV were decreased under conditions where decreased fat intake or malabsorption of nutrients may be present, such as in patients with chronic pancreatitis, malabsorption syndrome, acute hepatitis in the early stage, obstructive jaundice and in postoperative patients on total parenteral nutrition. On the other hand, the levels of apo A-IV were increased in patients with chronic renal failure and diabetes mellitus.

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Fourteen mouse monoclonal antibodies were produced in two fusions using hobo-human VLDL as antigen. On immunoblotting first with human VLDL and then with isolated human apo C-I, ten of the antibodies representing three isotypes, manifested specificity for apoprotein (apo) C-I. To assess whether the 10 antibodies were directed against the same or distinctively different epitopes of apo C-I, cross competition assays were performed wherein ¹²⁵I-monoclonal antibodies were made to compete vs. unlabeled antibodies for occupancy on immobilized VLDL-associated apo C-I. All antibodies cross competed to varying extents implying that they were directed against closely spaced epitopes. On immunoblotting with CNBr fragments, all of the epitopes were assigned to the CNBr I fragment of human apo C-I (amino acids 1-38).
A competitive solid-phase radioimmunoassay (RIA) was developed employing one of the anti-apo C-I antibodies. VLDL was adsorbed to plastic microtiter wells, and a limiting amount of the antibody was reacted with the adsorbed VLDL. The amount of monoclonal antibody that bound to the immobilized VLDL-apo C-I was determined with a ¹²⁵I-labeled goat anti-mouse IgG antibody. The addition of competitor apo C-I complexed with lipids resulted in reduced binding of the anti-apo C-I antibody to the immobilized VLDL-apo C-I. Competitor complexes consisted of an artificial lipid emulsion (Intralipid^R) incubated with apo C-I to yield phospholipid/apo C-I ratios of 1:1 to 75:1 (w/w). Increasing amounts of these complexes yielded competitive displacement curves of changing slopes, while isolated lipid-free apo C-I competed very poorly, suggesting that a conformation-dependent epitope was being probed. Other apoproteins (C-II, C-III, A-I, A-II and E) whether lipid-free or complexed with lipids did not compete.
The 40:1 (w/w) complex of Intralipid phospholipid/apo C-I was used as the primary assay standard because its displacement curve paralleled the curves obtained with whole plasma or isolated lipoproteins. Mean plasma concentration of apo C-I of 48 normolipidemic subjects was 25.0±11.7 (SD) mg/l. Plasma apo C-I contents correlated significantly with plasma triglyceride concentrations (n=93, r=+0.833, p<0.001).

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Most hypolipidemic drugs can not normalize plasma cholesterol and LDL levels in type II hyperlipoproteinemia (HLP) when used alone. Combinations of many of the hypolipidemic agents can have an additive effect. The bile acid sequestrants, cholestyramine, when given singly, can reduce total cholesterol values by 15 to 25% in patients with type II HLP. When combined with a variety of agents, plasma cholesterol concentration reduced by 20 to 45% and LDL-cholesterol by more than 50%. However, approximately 30 of the patients treated with cholestyramine complain unpalatability and gastrointestinal side effects.
Therefore, the search for a well-tolerated, palatable and effective hypocholesterolemic drug is worthwhile. The purpose of the present study was to investigate the effects of melinamide singly and in combination with probucol on plasma lipid, lipoprotein and apolipoprotein (apo) concentrations in primary type II hyperlipoproteinemia. Melinamide inhibit the absorption of cholesterol in intestinal mucosal cells mainly by inhibition of ACAT (acyl CoA cholesterolacyl transferase) activities. Eleven type II hyperlipoproteinemic patients (5 IIa, 6 IIb) were treated with melinamide (2, 250mg/day) for more than 16 weeks. Then probucol (750mg/day) was added for more than 16 weeks. Plasma concentrations of cholesterol (Ch), triglyceride (TG), HDL-Ch, apo A-I, A-II, B, C-II, C-III and E were measured.
Apolipoproteins were determined by single radial immunodiffusion method. Plasma cholesterol was reduced from 325mg/dl to 284mg/dl (12.7% reduction) by melinamide singly and further to 232mg/dl (18.3% further reduction) by combination therapy with probucol. Total reduction rate was 28.6% by combination therapy. (VLDL+LDL)-Ch was decreased from 269.4mg/dl to 229.7mg/dl by melinamide alone (14.8%) and further to 190.5mg/dl (17.1% further reduction) by combination therapy with probucol. Total reduction rate was 29.3% by combination therapy. HDL-Ch was not changed by melinamide singly but decreased from 55.4mg/dl to 41.2mg/dl by addition of probucol. Plasma TG did not show singificant changes. Plasma concentrations of apo B, C-II and C-III were decreased significantly by combination therapy with melinamide and probucol. The patient's compliance to this combination therapy was very well without side effects. The data in the present study indicated that combined treatment with melinamide and probucol for primary type II hyperlipoproteinemia is useful.

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Selectivity of LDL removal by double filtration (DF) and dextran-sulfate cellulose (DSC) column plasmapheresis (PP) was compared in six heterozygous familial hypercholesterolemic (FH) and a non-familial type IIb hyperlipoproteinemic patients. And, probucol, cholestyramine or CS 514, a newly developed inhibitor of HMG-Co A reductase was tested to elongate PP intervals. Two and half liters of plasma were treated at each PP. DFPP or DSC column PP was purformed alternatively to each patient. Probucol (1g/day), cholestyramine (12g/day) or CS 514 (10-20mg/day) was administered after PP without any combination. Plasma total cholesterol (TC), TG, VLDL-C, LDL-C, HDL-C and apoproteins A-I, A-II, B, C-II, C-III, E were measured serially. Lipoproteins were separated by ultracentrifugation and apoproteins were measured by RID method.
Sixty five percent of LDL-C, 45% of HDL-C, 43% of apo A-I, 65% of apo B, 56% of apo C-II, 54% of apo C-III and 75% of apo E were removed on average by DFPP with a second membrane filter of average pore diameter 300Å. The removal rate of LDL-C was significantly higher than that of HDL-C by DFPP. Fifty nine percent of LDL-C, 0% of HDL-C, 12% of apo A-I, 11% of apo A-II, 61% of apo B, 50% of apo C-II, 50% of apo C-III and 85% of apo E were removed on average by DSC column PP. Therefore, the selectivity of LDL removal was much better by DSC column PP than by DFPP. Plasma TC reached 250mg/dl within a week and 280mg/dl within 2 weeks without medication. On probucol treatment, it reached 250mg/dl between 13 and 16 days, and 280mg/dl between 26 and 41 days. TC reached 280mg/dl between 48 and 56 days on cholestyramine. On CS 514, it reached 250mg/dl between 8 days and days longer than 84, and reached 280mg/dl between 11 days and days longer than 84 days.
We concluded that plasma TC of heterozygous FH could be kept normal for long time by the combination therapy with PP and drugs.

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We investigated lipoprotein metabolism in fourteen patients with recessive X-linked ichthyosis (RXLI), a metabolic disease in which steroid sulfatase was deficient. Polyacrylamide gel electro-phoretic mobility of low density lipoprotein (LDL) was markedly increased in all patients. Plasma cholesterol (TC) levels ranged from normal to slightly low (mean±SD; 156±28mg/dl). In four patients, plasma triglyceride (TG) levels were slightly or moderately high which ranged from 150 to 365mg/dl. Plasma apoprotein B (apo B) levels were in normal range (99±17mg/dl). While apo B to TC ratios were higher than normal control (0.63±0.11 vs. 0.52±0.07, p<0.01).
In these patients, marked changes in lipid and apoprotein compositions of LDL fraction were observed, that is, esterified cholesterol content in LDL (LDL-CE) were significantly lower (37±4 vs. 41±2% of total lipids, p<0.01), in contrast, TG content (LDL-TG) were higher than in LDL fraction of control subjects (18±7 vs. 10±2, p<0.001), and apo B to cholesterol ratios in LDL were higher than those in normal control (1.21±0.19 vs. 0.73±0.05, p<0.001). These findings suggest that the rapidly migrating LDL in patients with RXLI is accompanied by remarkable changes of lipid and apoprotein compositions, which may related to increase of cholesterol sulfate in LDL fraction which has strong electronegativity.
This anionized LDL, in which cholesterol sulfate was increased, was detected to bind to the LDL receptor of fibroblasts, but the binding activity was slightly lower than normal LDL. This study shows that increase of cholesterol sulfate in LDL of patients with RXLI may influence LDL receptor affinity.

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There are so many papers which suggest that denatured lipoproteins involve in foam cell transformation. Acetylated LDL is frequently used for this purpose in vitro experiments. As far as natural occuring denatured lipoproteins concerned, cholesterol sulfate-rich and FFA-rich LDL are pointed out in this session. As aditional denatured lipoproteins, lipoproteins released from degraded foam cells and lipid deposited in extracellular matrix should be also taken into account.
Accumulation of cholesterol ester in foam cell after uptake of these denatured lipoproteins are discussed from two pathways; microsomal and lysosomal involvement.

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We investigated the relationship between the degree of arteriosclerosis in the coronary arteries and the malnutrition of cancerous persons. Arteriosclerotic lesions of the coronary arteries were found to be significantly less pronounced among persons dying from malignant tumors, especially in the case of cancer of the digestive system, other than in the case of nonmalignant diseases. Histologically, the diffuse or focal disappearance of Sudan III-stainable lipid in the coronary arteries of the cancerous persons, were observed and were found many foam cells, the increase of the smooth muscle cells, a large amount of glycosaminoglycans and new-formation of capillary in the luminal intima in the areas where the lipid disappeared.

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To study the effects of percutaneous transluminal angioplasty (PTCA), Transluminal coronary angioplasty (TCA) was performed on autopsied human hearts which consisted of 22 stenosed coronary arteries over than 50% of 15 cases. After postmortem coronary angiography (pressures 120mmHg), TCA was performed using the Simpson-Robert dilatation system (pressures 6 atm. 30sec twice). After second angiogram, stenotic lesions were quantified and analysed by histology with 121 serial step sections.
Results
1) Normal segment and mild atheromatous plaque can be dilated without histological change showing compression of atheromatous lesion.
2) The following complications were observed; 26% splitting of the intima at the transition of the normal segment to the plaque, 24% dissection between the intima and the media, 18% dissection of the media, 13% laceration of the intima and media, and 2% splitting near the atheroma. One of 22 arteries showed dissecting aneurysm.

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To evaluate the short-term efficacy of Percutaneous transluminal coronary angioplasty (PTCA), the results of follow-up coronary angiography in 101 patients with successful PTCA were analyzed. Coronary angiograms of 125 lesions after averaged 3.5 months follow-up were compared with those immediately after PTCA.
Of 125 lesions successfully dilated, restenosis was found in 27 lesions (22%). The incidence of restenosis was significantly higher in left anterior descending artery than in right coronary artery. It was also significantly higher in lesions with subtotal or total occlusion. 90 lesions showed neither restenosis nor improvement of the stenosis. 8 lesions (6%) revealed improvement of the stenosis as compared to the post-PTCA angiograms, in which the mean degree of stenosis reduced from 35±8% to 14±8%. No factors were associated with improvement of the stenosis. Of 8 lesions, 3 were infarct-related and 4 showed dissection immediately after PTCA. 3 lesions, however, were neither infarct-related nor dissected.
Improvement of the stenosis in a small number of lesions after relatively short-term follow-up indicated the possibility of the regression of coronary atherosclerosis initiated by PTCA. Further studies are needed to elucidate the long-term influence of PTCA on coronary atherosclerosis.

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The platelet derived growth factor (PDGF) is a powerful mitogen for cells of mesenchymal origin and a potent chemoattractrant for human neutrophils and for human monocytes. PDGF also activates neutrophils to aggregate, to adhere, to generate superoxide, and to release neutrophil specific ganule contents. The binding of human ¹²⁵I-PDGF to these cells was examined. Binding to neutrophils was nearly complete at 5min and to monocytes was complete at 30min. The K_d of ¹²⁵I-PDGF was 2nM for neutrophils and for monocytes; ∼2, 000 sites/neutrophil and/monocyte were found. Neither EGF nor FMLP were competitive with ¹²⁵I-PDGF for neutrophil binding. Unlabelled PDGF was fully competitive with ¹²⁵I-PDGF. Pre-incubation of PF4 and LA-PF4 at 10μg/ml blocks ¹²⁵I-PDGF binding to neutrophils. The K_d of ¹²⁵I-PDGF binding to neutrophils and monocytes correlates well with the optimal concentrations of PDGF for neutrophil and monocyte activation. These results suggest that PDGF interacts with specific high affinity receptor on the cell surface of neutrophils and monocytes, and that the receptor function might be regulated by PF4 and LA-PF4.

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Although PUFA have been reported to have an antiatherogenic effect, the mechanism is not yet fully understood. This paper investigated the direct actions of PUFA on the arterial SMC from the view point of growth control.
The effects of 12 PUFA on the proliferation of cultured SMC from rabbit thoracic aortas were studied in the growing condition. Arachidonic acid stimulated cell proliferation at low concentrations (<16.6μM) and inhibited it at higher concentrations (30-120μM). The stimulation was blocked by indomethacin. Drugs which stimulate prostaglandin synthesis from arachidonic acid in SMC also stimulated cell proliferatin which was again blocked by indomethacin. Exogenous PG E₂ stimulated cell proliferation at low concentrations. These results indicate that stimulation of cell proliferation by low concentration of arachidonic acid was mediated by prostaglandins synthesized from arachidonic acid in SMC. The inhibitory effects of high concentrations of arachidonic acid were blocked by 7 antioxidants tested which inhibit acyclic lipid peroxides synthesis from PUFA in SMC. Other PUFA than arachidonic acid all showed inhibitory effects on cell proliferation which were blocked by antioxidants. These results indicate that inhibition of cell proliferation by PUFA was mediated by acyclic lipid peroxides synthesized from PUFA in SMC.
Available PUFA contents (in phospholipids) such as arachidonic acid, di-homo-γ-linoleic acid, and adrenic acid were decreased in atheromatous lesions. Thinking of the results above, this decrease suggests that SMC rapidly proliferate in atheromatous lesions because SMC are released from growth control by PUFA. Thus PUFA may prevent the initiation and progression of atheroma formation through the control of cell proliferation.

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In order to study the process of intimal repair and its relationship to atherogenesis, the endothelial cells were isolated from human umbilical vein by collagenase digestion.
The endothelial cells were examined by immunostaining with antifactor VIII and antibody against fibronectin and stained with ruthenium red. Ultrastructurally fibronectin was localized in endoplasmic reticulum and basal region in endothelial cells grown on coverslips. Ruthenium red positive glycosaminoglycan was detected on the cell surface. The ³⁵S-sulfuric acid incorporation to determine of the synthesis of extracellular matrix in endothelium was enhanced by the conditioned medium of cultured smooth muscle cells. Endothelial cells may contribute to the extracellular matrix of the arterial wall as they regenerate after injury.
These findings suggest the important of fibronectin and glycosaminoglycan in the atherosclerosis.

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1) We studied the effect of heparin on the migration and proliferation of fetal bovine aortic EC and SMC in culture.
2) Heparin inhibited the proliferation of SMC and EC dose-dependently. Although Castellot et al. reported that the effect was specific for SMC, it was not specific for SMC in our study. It is not known how the difference between the results can be explained.
3) The inhibitory effect of heparin on SMC proliferation was neutralized partially by protamine sulfate.
4) Heparin specifically inhibited the migration of SMC dose-dependently, but not of EC.
5) Our results suggest that anti-atherogenecity of heparin might be due not only to the fully-understood anti-coagulatory and lipemia-clearing effect, but also to the inhibitory effect on the migration and proliferation of medial SMC.

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To evaluate the validity of Aortic Pulse Wave Velocity (PWV) as a non invasive method for diagnosis of atherosclerosis, we measured PWV for 231 Japanese diabetic subjects in Hiroshima and 522 Japanese-Americans in Hawaii, both are known to have high incidence of arteriosclerotic diseases.
In Hiroshima diabetics age, systolic blood pressure, duration of diabetes but not fasting blood sugar, HbA_1c, serum lipids and PWV were positively and significantly correlated. Diabetics with ischemic change of ECG, calcification of aorta observed in chest X-P and sclerotic change of retinal artery showed significantly higher PWV than those without.
In Japanese-Americans age, blood pressure, 2 hours blood glucose post 50g OGTT, uric acid but not obesity index, smoking habit, plasma cholesterol, triglyceride showed positive and significant correlation with PWV. Serum HDL-cholesterol was negatively correlated with PWV. It also appeared that sum of IRI post 50g glucose load which subdevided into five groups positively and significantly correlated with PWV. Subjects with arteriosclerotic diseases as determined by Rose's questionaire had significantly higher PWV than those without.
Thus PWV correlate many risk factors for atherosclerosis and is thought to be useful as non invasive diagnostic method for atherosclerosis. But it should be prudent to diagnose atherosclerosis only by PWV.

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Although a high prevalence of atherosclerotic diseases is observed in patients with diabetes mellitus, the precise mechanism has been unclear. In order to clarify the contribution of lipoprotein metabolism to the development of atherosclerosis in diabetes, we focussed on the metabolism of plasma remnant lipoproteins.
First, lipoprotein profiles in survivors of myocardial infarction (MI) were investigated. A high prevalence of mid-band in lipoprotein electro-phoresis, and increased cholesterol/triglyceride ratio in VLDL and elevated lipid contents in IDL were shown in them. These changes were seen exclusively in patients with MI whose glucose tolerance were impaired.
Second, lipoprotein profiles were also analysed in newly-diagnosed patients with diabetes or IGT, who has not yet suffered from atherosclerotic diseases. It revealed that IDL levels in patients with diabetes or IGT were higher than those in age- and sex-matched controls. Furthermore, 5 patients had elevated IDL levels to the extent of familial type 3 hyperlipoproteinemia. But phenotype of apolipoprotein E isoform of them were not E2/2 (E 3/3 in 2 cases, E4/3 in 2, and E 4/2 in 1). Thus, elevation of remnant lipoprotein was seen in diabetes, and plays an important role in occurrence of atherosclerotic diseases.
Finally, the mechanism of the remnant accumulation was investigated in experimental animal model. Streptozotocin-diabetic rats showed a marked hyperlipoproteinemia after an exogenous cholesterol load. The degree of elevation of remnants lipoproteins, especially that of chylomicron remnants, in diabetic rats fed a high-cholesterol diet was greatly higher than in non-diabetic rats fed it. When cholesterol-fed animals were treated with 17α-ethinyl estradiol or T₃, remnant lipoproteins were disappeared from plasma. These data suggest that some disorder exists in exogenous cholesterol transport in diabetes, and this disorder could be improved by the agents which are known to induce lipoprotein receptors.
In conclusion, increased remnant lipoprotein plays important role in coronary atherosclerotic disease in Japan, to which diabetes is closely related. The major mechanism of elevated remnant lipoprotein levels in diabetes might be explained by impaired exogenous cholesterol transport.

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Effects of insulin and growth factors on low-density lipoprotein (LDL) metabolism were studied in cultured human fibroblasts. Epidemal growth factor (EGF), fibroblast growth factor (FGF), and insulin increased LDL binding, internalization, and degradation in a serum free system by approximately 39%, 25%, and 75% respectively. Both EGF and FGF had additive effects on the insulin stimulated LDL metabolism in the cells. In the presence of 2mM hydroxyurea an inhibitor of DNA synthesis, basal acivities of LDL pathway were declined, however the insulin stimulation was not altered. Insulin also stimulated (U-¹⁴C) D-glucose incorporation into the same fibroblasts, and the half maximal effects were observed at the insulin concentration of 5ng/m/. Dose-response curves of glucose incorporation to insulin were similar to those of LDL pathway to insulin.
Results indicate that the stimulatory effects of insulin on LDL pathway is independent of cell proliferation, and also suggest that insulin plays a physiological role in LDL metabolism comparable to that in carbohydrate metabolism.

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Platelets contain major growth factors. PDGF stimulates the proliferation of vascular smooth muscle cells (VSMC) and fibroblasts, which is an early event in the pathogenesis of atherosclerosis. We studied on the growth promoting activity (GPA) in platelet supernatant (PS) obtained from human diabetics with atherosclerotic lesions or streptozotocin-induced diabetic rats (STZ). PS was prepared either by freeze-thawing in insulin-dependent diabetic patients (IDDM) or STZ and by platelet aggregation with collagen in noninsulin-dependent diabetics (NIDDM). GPA was measured as an incorporation of ³H-thymidine into DNA following the addition of PS on cultured human or rat VSMC, 3T3 cells, and rat lung fibroblasts. As results, PS with the addition of plasma derived serum stimulated DNA synthesis of cultured VSMC and fibroblasts in a dose dependent manner. GPA on cultured VSMC and fibroblasts was significantly increased in PS from IDDM as compared with age matched controls. Furthermore, this enhanced GPA in PS from IDDM was normalized by chronic intensive insulin therapy. It was also observed that platelet aggregation was significantly increased and GPA enhanced in PS following platelet aggregation in NIDDM. From these results, it is suggested that enhanced GPA in platelets can cause the atherogenesis in human diabetics. The examination of GPA can be useful for the clinical evaluation of diabetic vascular complications. On the other hand, GPA in PS from STZ was not significantly different from control rats. The role of GPA in PS between different species is needed to be clearr in the future study.

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We studied the effects of nifedipine, calcium channel blocker, on atherosclerosis of cholesterolfed rabbits and Watanabe heritable hyperlipidemic rabbits (WHHL rabbits). In experiments, rabbits were divided into two groups. Nifedipine was given 20mg orally twice a day (nifedipine group), and in control group placebo was given in the same way (placebo group). In cholesterol-fed rabbits, the percentage of aortic intimal surface area coverd by atherosclerotic lesions (% AS) was 25.9±7.6 (mean±s. d.) in nifedipine group (n=7), and 55.6±22.8% in placebo group (n=8) (p<0.01). In WHHL rabbits, %AS was 33.4±14.1% in nifedipine group (n=5), and 27.0±11.7% in placebo group (n=6) (n. s.). We concluded that nifedipine suppressed atherosclerosis in cholesterol-fed rabbits but not in WHHL rabbits.
To elucidate the mechanism of anti-atherosclerotic effect of calcium channel blocker, we studied the effects of nifedipine on cholesterol metabolism in cultures rabbit skin fibroblasts. Nifedipine enhanced cholesterol accumulation during incubation with hyperlipemic rabbit serum. Also, nifedipine retarded cholesterol efflux. In these experimental conditions, we could not validate the anti-atherosclerotic action of nifedipine. The further studies are required.

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We studied the effect of diltiazem and theophylline on bovine aortic smooth muscle cells (SMC) and endotherial cells (EC) in culture. The SMC which were cultured with 10^-4M diltiazem or 10^-3M theophylline reached the stationary phase at lower density than without them. And they showed polygonal endotherial cell-like formes, keeping a cobble stone-like monolayer. Under these conditions, Factor VIII related antigen was proved by immunofluolescent staining. When the medium was changed into the medium without diltiazem (or theophylline), the SMC recovered the usual state of SMC showing spindle-shaped multilayerd proliferation and had negative Factor VIII related antigen. Therefore, these appeared to be a reversible change.
Transmission electron microscopic (TEM) examination of the SMC which were cultured with 10^-4M diltiazem revealed that vacuolar degeneration was present in them, that mitochondria, rough endoplasmic reticula and microfiraments in them had a tendency to decrease in quantity, that the area of contact surfaces between each cell was less than that of control endtherial cells and that there was no tight junction at contact surfaces. And then, TEM examination of SMC cultured with 10^-3M theophylline showed the similar changes with that with 10^-4 diltiazem, but 10^-3 theophylline did not make mitochondria and rough endoplasmic reticula degeneration.

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We have shown that marked reduction of serum cholesterol and marked regression of cutaneous and tendon xanthomas were obtained by probucol treatment even in homozygous as well as heterozygous cases of familial hypercholesterolemia under low fat and low calory diet, although the treatment caused a remarkable decrease in HDL-cholesterol (Atherosclerosis 48, 157, 1983). In order to elucidate the mechanism of the effects of probucol, we investigated the relation between the extent of the regression of lipid storage evaluated by xeroradiography of Achilles tendon thickness and the changes in lipoproteins. The reduction of Achilles tendon thickness was not correlate with that of serum cholesterol, while it correlated closely with the extent of the reduction of HDL-cholesterol. High performance liquid chromatography of lipoproteins revealed that HDL₂ particles from probucol-treated cases with familial hypercholesterolemia were small in size. In addition, net transfer rate of cholesteryl ester from HDL to larger particles was found to be enhanced by probucol treatment. These findings provide us the hypotheses that probucol may enhance cholesterol catabolism through the other pathway but LDL-receptors by stimulating the cholesterol transfer from HDL to larger lipoprotein particles and consequently may enhance the reverse transfer of Ch to take lipids out from the tissues.

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Hypertriacylglycerolemia is often accompanied by low level of plasma high density lipoprotein (HDL). In order to determine whether or not HDL can be a regulartory factor of very low density lipoprotein (VLDL) metabolism, we studied the effect of HDL on VLDL-triacylglycerol (TG) hydrolysis by lipoprotein lipase (LpL) in the presence and absence of d>1.25 fraction. HDL enhanced VLDL-TG hydrolysis by bovine milk LpL; low density lipoprotein (LDL) did not promote the hydrolysis. The increased hydrolysis of VLDL-TG by HDL further enhanced by d>1.25 fraction. Though d>1.25 fraction also had a stimulatory effect on VLDL-TG hydrolysis apart from the effect of HDL, the VLDL-TG was doubly increased by simultaneous existence of HDL and d>1.25 fraction. HDL did not show any effect on the hydrolysis of Triton X-100-emulsified TG. d>1.25 fraction had a weak stimulatory effect on the detergent-emulsified TG. This enhanced VLDL-TG hydrolysis by HDL was not due to additional apoprotein C-II (apo C-II) contained in HDL, because the VLDL hydrolysis system used in the experiment contained a sufficient amount of apo C-II in VLDL and further addition of apo C-II did not increase the hydrolysis. Phosphatidylcholine (PC) liposome increased VLDL-TG hydrolysis by LpL. On phenyl-Sepharose chromatography of d>1.25 fraction showed that VLDL-TG hydrolysis stimulatory fraction was coeluted with cholesteryl ester (CE) transfer activity. Triton X-100-emulsified TG hydrolysis by LpL was inhibited as the content of CE in the TG emulsion was increased.
These results suggest that low HDL level in plasma may retard VLDL hydrolysis and cause hypertriacylglycerolemia. PC liposome also activated LpL in VLDL-TG hydrolysis. HDL is a phospholipid rich lipoprotein, so it might act as a liposome in plasma. d>1.25 fraction seemed to play an important role in VLDL hydrolysis. The fact that LpL was inhibited by CE and VLDL hydrolysis stimulatory fraction was coeluted with CE transfer activity on the hydrophobic chromatography implied that d>1.25 fraction may transfer CE from VLDL or VLDL remnant to HDL during the hydrolysis by LpL to promote TG hydrolysis of VLDL and VLDL remnant. The mechanism by which HDL and d>1.25 fraction promoted VLDL-TG hydrolysis by LpL remains to be clarified.

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We have reported that H-35 rat hepatoma cell line has at least three different lipoprotein receptors for LDL, chylomicron-remnants (CM-R) and HDL. Aim of the present study was to investigate the effect of cell density on regulation of lipoprotein receptors.
The specific binding of ¹²⁵I-LDL was greater at all cell densities when cells were grown in lipoprotein deficient serum (LPDS) rather than in new born calf serum (NBCS). Cellular binding activity was affected by cell density to a much greater extent in LPDS than in NBCS. Binding of ¹²⁵I-non-apo E-HDL was increased only at low cell density whether cells were grown in LPDS or NBCS. There were no appreciable differences in binding activity between LPDS and NBCS. Binding of ¹²⁵I-CM-R appeared to be increased in LPDS at all cell densities, but cell density per se showed little consistent effect on binding.
Thus, the three lipoprotein binding activities of H-35 cell were uniquely affected by cell density.

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It is reported that calcium antagonists have an antiatherosclerotic effect by some investigators. But the mechanism of the effect is obscure until now. As one of the mechanism of the antiatherosclerotic effect of calcium antagonist is reported that there is a stimuratory effect on PGI₂ production of arterial smooth muscle cells (SMCs) and endothelial cells. We also reported that some kinds of calcium antagonist had a stimulatory effect on PGI₂ release from cultured rat aortic smooth muscle cells. To study the mechanism of the effect of calcium antagonist, we investigated the motion of calcium in or extra SMCs.
The method for culture of SMCs and the assay of PGI₂ was the same as our previous report.
To measure the calcium concentration we used Atomic Absorption method.
As conclusion;
1) Calcium in the medium tended to stimulate the growth of arterial SMCs in culture.
2) Positive corelation with calcium concentration in the medium and the calcium content of intra SMCs in culture was observed.
3) Calcium ionophore, A23187, increased calcium content of intra SMCs in culture.
4) Calcium in the medium significantly increased the release of PGI₂ into the medium in a dose-related manner.
5) A23187 significantly increased the release of PGI₂ into the medium dose-dependently.

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It has been suggested that increased residence time of LDL in the plasma may lead to change of this lipoprotein class allowing them to be taken up by macrophage. The significance of prolonged residence time for LDL-cell interaction remains still speculative. Earlier workers have reported that they considered to exist structural changes of LDL in familial hypercholesterolemia (FH). They showed that LDL in FH was cholesterol-enriched and triglyceride-poor. However, recent studies have shown the possibility that LDL in FH is cholesterol-poor rather than controls. Then, we were investigated that the effects of hypercholesterolemia in 11 FH (hetero, Type IIa) subjects. Plasma lipoprotein were separated by ultracentrifugation. Lipoprotein cholesterol, triglyceride and phospholipid were measured by enzymatic procedure. The lipid composition and phospholipid composition were determined by thin layer chromatography.
The results as follows:
1) In FH subjects, serum total cholesterol (TC), phospholipid (PL) and LDL were significantly higher than controls.
2) In the lipid composition VLDL and HDL contained higher amounts of cholesterol ester (CE) than those in the controls. Contrarily, LDL had less amounts of CE than in the controls.
3) In the phospholipid compositions, a decrease in Sphingomyelin (SM) and an increase in Phosphatidyl choline (PC) were observed in LDL. Contrarily, an increase in SM and a decrease in PC were observed in HDL. On the other hand, there were not any changes in VLDL.
4) In the levels of serum apolipoproteins, FH patients had higher apo A-I and A-II levels than those in the controls.
Discussion and conclusion
It is understood that the biomembrane increase fluidity when PC and PE increase and SM decrease in PL composition. Depleted FC in lipid composition also increase the membrane fluidity. And it might be thought that lipoprotein particles become smaller in size if core components is less as compared with surface components. In these respects, above mentioned data may indicate that LDL particles become smaller and increase fluidity in FH. Furthermore, the data indicate that HDL may become larger particles enriched in CE.

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The aim of this study is to ascertain the anti-atherosclerotic effects of elastase to the coronary atherosclerosis of spontaneously diabetic GK (Goto-Kakizaki) rat fed with an atherogenic diet. GK rats were fed with: 1) an atherogenic diet (2% cholesterol, 0.5% cholic acid, 10% lard, 5% cane sugar, 0.2% methylthiouracil, and 82.3% basal diet) for 6 weeks with 35, 000 units/kg body weight of vitamin D₂ for initial 4 days (athero-sclerosis group), 2) an atherogenic diet plus elastase injection of 450 ELU/kg/day for 6 weeks (EL group), and 3) standard chow for 6 weeks (control group). After the completion of the experiment, histological studies were performed on coronary arteries obtained from three groups. Oral glucose tolerance test (GTT, 2g/kg body weight) revealed no statistically significant differences between at pre and post 6 weeks of the experiment in all three groups. Histological studies showed normal coronaries in control group. However, coronaries in atherosclerosis group displayed remarkable atheromatous changes characterized with marked destruction and loss of elastic fibers, calcification, and accumulation of foam cells. In EL group, on the other hand, there were fewer foam cells, less prominent calcification, and elastic fibers were preserved considerably. Morphometrical analyses of coronary arteries revealed that ratio of internal lumen was significantly narrower, relative wall thickness was larger in atherosclerosis group as compared with control group and EL group (p<0.05). On the other hand, There were no significant differences in these parameters between control group and EL group. Foam cell occupancy rate was significantly smaller in EL group than that of atherosclerosis group (p<0.01). These data suggests that elastase exhibits an inhibitory effect on the coronary atherosclerosis in diabetic GK rats.

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The effect of pantethine on an ultrastructural changes of mitochondria membrane in brain microvessels of spontaneously hypertensive rats (SHR) was examined in connection with restoration of decrease in the activity of fatty acid oxidation in the organ in vivo. SHR of 15 weeks old were treated with pantethine at a dose of 300mg/kg/day for 4 weeks. Then, the samples of their brain microvessels were removed to compare the lesions of smooth muscle cells with that of non-treated SHR of the same age.
In SHR aged from 8 to 19 weeks, electron micro-scopic observation of brain microvessels revealed the presence of damaged mitochondria in smooth muscle cells. Changes in mitochondria structure of SHR of 8 weeks old are enlargement in size, increase in electron density and projection of some external membrane. Moreover, the fragmentation of cristae in enlarged mitochondria and disappearance of mitochondria structure were observed in SHR of 19 weeks old.
In contrast to a marked changes in mitochondria, a significant changes in lysosomes of smooth muscle cells was not observed in SHR aged from 8 to 19 weeks at all.
Pantethine treatment substantially restored an advanced damage of mitochondria structure to those lesions observed in SHR of 8 weeks old. However, this drug did not affect blood pressure of SHR at all.
From these findings, we speculated that the damages of mitochondria structure in smooth muscle cells were restored by the pantethine treatment, especially through the stabilization of mitochondria membrane. These actions of pantethine may contribute to maintain the activity of fatty acid oxidation in mitochondria.

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Serum levels of lipids (triglyceride: TG, total cholesterol: TC and HDL cholesterol: HDL-C) and apolipoproteins (Apo A-I, A-II, B, C-II, C-III and E) were measured in 76 survivors of cerebral infarction (CI) and in 57 age-matched healthy controls. TG and TC levels were determined enzymatically and HDL-C level was determined by the heparin-Ca method. Apolipoproteins were measured by a single radial immunodiffusion method (Apoplate, Daiichi Chemicals : Tokyo).
TG level was significantly higher and HDL-C level was significantly lower in the CI group than in the controls although there was no significant difference in TC level.
Apo A-I level was not so decreased as expected from the decrease of HDL-C level and then the HDL-C/Apo A-I ratio was significantly lower in the CI group. The value of this ratio was negatively correlated with the TG level. We suggest that cholesterol ester is excessively transferred from HDL to VLDL during the disturbed catabolism of VLDL in CI patients.
Apo B level was significantly higher in the CI group than in the control group but the (TC-HDL-C)/Apo B ratio was not different between the two groups.
The levels of Apo C-II, Apo C-III and Apo E tended to be higher in the CI group but not significantly.

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