Elastin substructure, sequential changes of elastolysis were studied by electron microscopy using tannic acid stain. Prolonged treatment of rabbit carotid arteries with periodate or osmium tetroxide removed tannic acid-reactive materials from the central amorphous portion of elastic fibers with resultant disclosure of the fibrils approximately 4nm in diameter, which were readily digested by elastase. Based on these findings it was considered that the central amorphous portion consists of a fibrillar network of elastin which is embedded in tannic acid-reactive materials. In the stenotic segments induced by constriction of the common carotid arteries of rabbits, elastolysis proceeded through two steps: initially tannic acidreactive materials were removed, and then fibrillar network of elastin dissolved remaining peripheral microfibrils.
Light and electron microscopic observations were made on elastolysis and elastogenesis during the developement of experimental atherosclerosis produced in cholesterol-fed rabbits by partially constricting the carotid artery. The internal elastic lamina disrupted within a week after constriction in the same process of elastolysis observed in the stenotic segments. During this time, smooth muscle cells were migrated from the media into the intima, where they proliferated. Subsequently numerous elastic fibers were formed by smooth muscle cells and the disrupted internal elastic lamina was also regenerated. In advanced stages, elastolysis predominated over elastogenesis and elastic fibers deposited in the intima were degraded showing the similar features to those observed in the stenotic segments. Whereas, bundles of microfibrils lacking elastin or associated with only tiny elastin aggregates were seen deposited around the smooth muscle cells. Large amount of lipids deposited particularly in the central portion of the elastic fibers and calcium deposited on microfibrils, suggesting that degraded elastin and overproduced microfibrils may have predilection for the deposition of lipids and calcium.

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Smooth muscle cells, fibroblasts derived from various kinds of fibers, chondrocytes originating in elastic cartilage and neutrophils are known as cells that produce elastin and repair damaged elastic fibers or regenerate them. It has also been reported that an elastin-like protein is formed from endothelial cell culture. These facts suggest that various factors are involved in the biosynthesis of elastin.
Using the SD-SLC rat, an atherosclerosis model was prepared administering an atherogenic diet and vitamin D₂ in order to determine the effects of elastase on the development, progression and regression of atherosclerosis.
At 6 and 9 weeks after initiation of study, the animals were sacrificed and their thoracic and abdominal aortas removed. The media was isolated by the explant method, and a tissue culture was done while determining tissue levels of elastin. Considering that smooth muscle cells from the control group were more active in division, proliferation and swarming than those from the treated group, it seems to stand to reason to assume that biological kinetics of smooth muscle cells differ from the control group to the treated.

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In the regression of atherosclerosis of rabbit common carotid artery caused by combination of cholesterol feeding and arterial freeze injury, early cessation of cholesterol feeding induced marked reverse of the lesion, but the reversibility at the late cessation of cholesterol feeding was bad.
Electron microscopically the intima showed the regression of atherosclerosis, muscle cells were synthetic phenotype at the early lesion and they turned to contractile phenotype in the late lesion. Elastic fibers of the regressed intima were abundant in the luminal intima and small in number in the deep intima, but however, collagen fibers of the intima were abundant in the deep intima and the amount of them decreased in the luminal side.

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Experimental atherosclerosis was induced in rats by administering Vit. D₂ and atherogenic diet in order to examine the relation between metabolism of aortic elastin and lipid deposition.
It was found as the result that 1) Relative amount of total cholesterol (free and ester) and C_18:1/C_18:2 in elastin fr. of the aorta was tend to increase. On the other hand, relative amounts of triglyceride and phospholipid in the elastin fr, were tend to decrease more in the induced atherosclerotic aorta than in the control one. 2) Residual radioactivity (³H) in urine, serum and aortic elastin fr, in the induced atherosclerotic rats after administering 27.8μCi/rat/day of ³H-Lys for 10 days, was examined, and it was found that the radioactivity was tend to be higher in the atherosclerotic rats than in the control one, which might reflect the fact that the turnover of elastin was decreased more in the atherosclerotic rats. 3) Urinary desmosine, as determined by radioimmunoassay in atherosclerotic rats was tend to be lower than in the control rats at various stages (in 2, 4, 6, 8, 10 weeks). It may be indicated that the turnover of elastin was slower in the atherosclerotic rats than in the normal ones. 4) Externally administered porcine pancreatic elastase preparation might exert such improving effects as stated above (especially the activation of elastin turnover, presumably resulting from the accelated degradation of elastin). The present data suggested that abnormal metabolism of elastin could be one of the factors of lipid deposition in atherosclerosis.

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We studied effects of long term treatment of four cholesterol lowering drugs; compactin, cholestylamine, probucol and niceritorol on lipid and lipoprotein concentration and on the incidence of coronary heart disease in 111 patients with familial hypercholesterolemia (FH).
1. In 4 patients with homozygous FH, mean serum cholesterol levels decreased by 24 per cent with drug treatment of large dose of compactin in one patient and of 1-1.5g/day probucol in the other three patients. One patient who was suffered from myocardial infarction before treatment died of heart attack during follow-up period. Other three patients were performed AC bypass operation and their anginal attacks were controled well.
2. In heterozygous patients, mean serum cholesterol levels decreased by 19 per cent. Three drugs which are known to effective for FH decreased cholesterol levels by -26±11%, -13±8% and -15±7%, resspectively. In addition, niceritorol was also effective for heterozygous FH when the sufficient doses could be given without side effect such as flashing. More than 5 per cent decrease in Achilles tendon thickness (ATT) was found in one-third patients. Although the regression of ATT required the reduction of serum cholesterol level, the reduction of ATT and that of serum cholesterol level was not closely correlated. In the patients treated with probucol, the regression of ATT was correlated with the reduction of HDL-cholesterol level.
3. During follow-up period, three patients suffered of fatal or nonfatal myocardial infarction. All three patients had had manifest coronary artery disease at entry. The patients without coronary artery disease at entry did not suffered from new cardiac events during the period. Even if the patients had coronary artery disease at entry, combination therapy of surgery with hypolipidemic drugs could improve their prognosis.
In conclusion, assessment of coronary arterial lesion and treatment with cholesterol-lowering drugs might be needed before the manifestation of coronary heart disease in patients with FH.

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To elucidate the relationship between vasoconstrictive hormones and prostaglandin (PG) sunthesis in vascular smooth muscle cells (VSMC), the effects of 8-arginine-vasopressin (AVP) and angiotensin II (ANG II) on PG synthesis in cultured VSMC from rat mesenteric artery was examined. Vasoconstrictive hormones as well as arachidonic acid and calcium ionophore A23187, stimulated PG synthesis (6-keto-PGF_1α>>PGF_2α>PGE₂, TXB₂), which was completely inhibited by acetylsalicylic acid. Vasoconstrictive hormone-evoked 6-keto-PGF_1α synthesis was abolished by the depletion of extracellular calcium. The hormonal or non-hormonal stimulation of 6-keto-PGF_1α production was observed in cells up to the 24th passage (162 days after the isolation). These data suggest that vasopressin and angiotensin II stimulate prostacyclin synthesis in VSMC, which may modulate the vasoconstrictive action of these hormones.

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The vascular endothelium is known to act as a selective barrier for macromolecules between the intra- and extra- vascular lumen. To clarify the molecular mechanism of transport through endothelial cells, we developed an in vitro model system, porcine arterial endothelial cells cultured on type I collagen gel supported by dacron sheet. In this study, the relation between molecular weight of fluorescein dextran (FD) and transport through endothelial barrier was studied.
The endothelial cell monolayer supported by dacron sheet was set between two separate chambers, and appropriate amounts of FD (molecular weight; 4K, 10K, 20K, 70K, 150K) were introduced into upper compartment, FD in lower compartment thereafter was determined by fluorescence intensity.
Scanning and transmission electromicroscopy showed that the endothelial cells were confluent 2-3 days after seeding of the cells. The permeation rates of FD through the monolayer were dependent on the molecular weight.

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In order to understand a mechanism of monocyte-endothelial cell interactions, the present study was performed to investigate the degradation of sulfated proteoglycans in the subendothelial extracellular matrix (ECM) by monocytemacrophages in vitro. Adherent mononuclear leucocytes isolated from human peripheral blood were plated on the [³⁵S]O₄-prelabeled ECM coated dishes prepared from cultured porcine aortic endothelial cells. The nature of radiolabeled materials released upon incubation of the ECM with cells at 37°C for 48-72hrs was studied by applying the culture medium to gel filtration on Sepharose CL-6B columns. The monocyte-macrophages solubilized the labeled matrix to a lower mol, wt. component compared with the material released during exposure of the ECM to medium without cells that eluted with the void volume. Degradation products eluted in the lower mol. wt. peak were precipitated with cetylpyridinium chloride and were resistant to further digestion with alkali, pronase or chondroitin ABC lyase, but were converted to further lower mol, wt. fragments after nitrous acid or heparitinase treatment. The size of intact glycosaminoglycan side chains determined by subjecting the ECM to cleavage with alkali or pronase was larger than that of degradation products released by the cells. Heparin inhibited the degrading activity of cells.
These results suggest that the attachment and subsequent invasion of vascular endothelium by monocyte-macrophages may involve the production of ECM heparan sulfate degrading activity by these cells.

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To elucidate the mechanism of cholesterol accumulation in cultured cells under hypoxic conditions which we previously reported, the effect of hypoxia on the efflux of cholesterol from cultured rabbit skin fibroblasts was tested.
Cells were incubated with hyperlipemic rabbit serum (HRS) for 48 hours (load phase), then washed three times with phosphate buffered saline and incubated with normolipemic rabbit serum (NRS) for 24 hours (efflux phase). In the efflux phase cells were divided into two groups, one group was incubated under 95% N₂ and 5% CO₂ (hypoxic group), another was 95% room air and 5% CO₂ (control group).
At the end of load phase cellular content of esterified cholesterol was 39.0±1.5nmol/mg protein and after efflux phase it was remarkably decreased in control group (22.4±2.5nmol/mg protein) while it was almost unchanged in hypoxic group (38.2±1.7nmol/mg protein).
When cells were incubated with lipoprotein deficient serum instead of NRS to exclude the uptake of extracellular cholesterol in efflux phase, the cellular content of esterified cholesterol was also significantly higher in hypoxic group than control group (53.1±1.5, 36.2±3.2nmol/mg protein respectively).
These results suggested that the affected efflux may play a role in accumulation of esterified cholesterol in cells under hypoxic conditions.

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For more than 30 years, we reported that experimental atherosclerosis similar to that of human beings was produced in monkeys by vitamin B₆ deprived diet. However, the effect of vitamin B₆ deficiency on forming atherosclerotic lesion was not obvious. By using vitamin B₆ antagonists, we examined the effect of vitamin B₆ deficiency on the migration and proliferation of the cultured endothelial cells from fetal bovine aorta. We also examined the effect of vitamin B₆ itself. The patients with homocystinuria, who lack the activity of cystathionine synthetase which needs vitamin B₆ as a cofactor, have the tendency to suffer from atherosclerosis in their early life. So it was considered that the impairment of sulfur-containing amino acid metabolism might play a role in the formation of atherosclerosis in monkeys fed on vitamin B₆ deprived diet. Because homocysteine in the blood was increased in the patients with cystathionine synthetase deficiency, we also studied the effect of homocysteine.
We evaluated the effect of the above substances on the cultured endothelial cells with cell migration assay. At this assay, when the seeded endothelial cells had formed the cell monolayer in a tissue culture dish, we removed half of the monolayer sheet with a rubber policeman. One week later, we measured the area of outgrowth portion (repaired area) from the edge of the remaining monolayer sheet. At first, vitamin B₆ antagonists, deoxypyridoxine and isoniazid, were tested. Deoxypyridoxine suppressed the repair from the edge significantly at concentration 5mM. Isoniazid suppressed the repair significantly at concentration 0.1mM. We also evaluated the effect of vitamin B₆ itself. Pyridoxal phosphate and pyridoxal HCl demonstrated no difference between control and concentration 0.01mM, 0.1mM but suppressed completely at concentration 1mM. Then vitamin B₆ and its antagonist were added to test media simultaneously. When deoxypyridoxine 5mM and pyridoxal phosphate 0.1mM were added, the repairing was accelerated. Homocysteine accelerated the repair significantly at 2mM but suppressed at 5 and 10mM. Isoniazid inhibited growth and migration more strongly than deoxypyridoxine. This difference might be according to the difference of mechanism as an antagonist. The suppressive effect of deoxypyridoxine was counteracted by addition of vitamin B₆. It was known that homocysteine had endothelial cell detaching potential and increased the number of circulating endothelial cells when it was injected into experimental animals. We expected that homocysteine would suppress the repair but it accelerated the repair at lower concentrations but suppressed at higher concentrations. The role of vitamin B₆ deficiency in the formation of atherosclerosis is not clear in this study, which uses endothelial cells alone in vitro. It was supposed that the atherosclerosis by vitamin B₆ depletion might be caused by the impairment of multiple factors, for example, endothelial cells, smooth muscle cells, platelets, coagulants and their interactions in vivo.

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The effects of phospholipid fatty acid modification on the esterification of cholesterol was studied in rabbit cultured aortic smooth muscle cells (SMC). When confluent SMC were incubated with LDL for 16hrs, saturated fatty acids such as palmitic acid and stearic acid and monoen fatty acid (oleic acid) were increased and arachidonic acid was decreased in the phospholipid of SMC. When SMC was incubated with palmitic acid (60μM), palmitic acid was increased in the phospholipid. With arachidonic acid, arachidonic acid and adrenic acid were increased and oleic acid was decreased. These changes were similar to those seen in the atheromatous lesions.
Pretreatment of SMC with palmitic acid or arachidonic acid mentioned above increased or decreased the esterification of cholesterol in a dosedependent manner, respectively. Moreover, the reaction was increased by oleic acid, not affected by linoleic acid and decreased by linolenic, eicosapentaenoic and dochosahexaenoic acids. Pretreatment with LDL dose-dependently increased the reaction, which was suppressed by the concurrent treatment with arachidonic acid.
These results suggest that cholesterol esterification in microsomes was regulated by membrane phospholipids which are in turn affected by fatty acid composition. The possible mechanism of stimulatory effect of LDL on cholesterol esterification was discussed from the view point of fatty acid modification of phospholipids.

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The cholesterol amount of SMC was significantly decreased in SHRSP and stroke-resistant SHR (SHRSR) compared with normotensive Wistar-kyoto rats (WKY).
Incorporation of [¹⁴C] acetate into cholesterol in SMC was significantly decreased in SHRSP compared with WKY. In SHRSR the cholesterol synthesis tended to decrease, but there was no significant difference between WKY and SHRSR.
These results suggest that SHRSP have a genetic abnormalities in cholesterol synthesis system. Cholesterol in the membranes regulates phospholipid fluidity. These abnormalities may introduce diverse membrane abnormalities which may be partly involved in the mechanisms of hypertension and related to vascular lesions in SHRSP.

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1) We studied the erect of heparin and polyran T, a dextran sulfate, on the proliferation and migration of arterial smooth muscle cells in culture.
2) Heparin inhibited the migration and proliferation of smooth muscle cells dose-dependently.
3) The inhibitory effect of heparin on smooth muscle cell migration was partially antagonized by protamine sulfate.
4) Polyran T had a similar effect on the proliferation and migration of smooth muscle cells.
5) The present study suggests that anti-atherogenicity of heparin might be not only due to the anti-coagulative and lipemia-clearing effect, but also due to the inhibitory effect on the migration and proliferation of arterial smooth muscle cells.

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To clarify the mechanism of protection from atherosclerosis by calcium antagonist, we examined the effect of diltiazem on damages of cultured aortic smooth muscle cells (CASM) induced by hyperlipidemic serum (HLS). CASM were prepared according to the modified method of Chamley et al. After 7 days of cultivation, the concentration of the medium containing FBS was reduced gradually to stop the proliferation. Then the medium was replaced with the medium containing HLS or HLS and diltiazem (1μg/ml). HLS showed injurious action on the CASM. The trypan blue positive cells (dead cells) were 14±3% in control, 22±2% in HLS and 15±4% in HLS plus diltiazem. The ⁵¹Cr-%release was 10±1% in control, 16±1% in HLS and 14±1% in HLS plus diltiazem. The ⁴⁵Ca activity of CASM was significantly increased by adding HLS, while this elevated ⁴⁵Ca activity was significantly reduced by adding diltiazem.
Based on these data, we conclude that diltiazem suppresses HLS-induced necrosis of cultured aortic smooth muscle cells by reducing intracellular calcium. This mechanism might play an important role in the protection of atherosclerosis by the calcium antagonists.

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1) We devised a method to assess the migratory activity of cells in culture and studied the mechanism of cultured vascular endothelial and smooth muscle cell migration.
2) The present results suggest that cyclic nucleotides might mediate the migratory activity of the cells and that the cytoskeletal system including microfilaments and microtubules would be essential in the migration.
3) The participation of Ca²⁺-calmodulin system was not demonstrated in this study, although low concentration of extracellular Ca²⁺ was essential. 4) 12-HETE had no effect on the migration of endothelial and smooth muscle cells.

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Wistar rats were placed in an orthostatic state 3 hours every day for 11 months.
Arteriosclerotic intimal thickening of the small coronary arteries accompanied by small infarctions of the myocardium were detected. Neither necrotic nor PN-like lesions were seen.
Blood pressure increased gradually and showed levels of 180mmHg at the end of the experiment.
Further studies are necessary to determine the cause of intimal thickening of coronary arteries, that is, whether it derives from orthostatic circulation or from a hypertensive condition following orthostatic experiment.

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The role of superoxide produced by phorbol myristate stimulated human monocyte in the oxidation of low density lipoprotein (LDL) was examined. The modification of LDL was measured by both substance that react with thiobarbituric acid and electrophoretic mobility of the lipoprotein after incubation of LDL and mononuclear cells containing monocytes under the stimulation of PMA.
Monocytes mediated LDL oxidation was time and cell number dependent. The modification of LDL was completely abolished when superoxide dismutase (SOD) was added. The monocytes from patients with genetic deficiencies of superoxide generation (chronic granulomatous disease) failed to modify LDL. These findings suggest that the mediator of LDL oxidation by human blood mononuclear cells is superoxide.
LDL oxidation was observed to progress even after the cells had ceased to generate superoxide. The addition of SOD 4 hours after stimulation of the cells with PMA partially inhibited LDL oxidation, while later addition of SOD has little inhibitory effect.
Thus superoxide is able to initiate the LDL oxidation although be independent of the progression of process of LDL oxidation.

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Carboxyl esterase which essentially hidrolyzes short chain fatty acids can hydrolyze triolein in the presence of triolein hydrolysis promoting factor (THPF) in serum.
To evaluate the effect of carboxyl esterase on very low density lipoprotein (VLDL) metabolism in smooth muscle cells and macrophages, the changes of lipid contents in these cells after incubation with carboxyl esterase, THPF and VLDL were determined.
In smooth muscle cells, when VLDL was added in the presence of carboxyl esterase and THPF, triglyceride content was increased more than cholesterol ester content. Whereas, in macrophages, cholesterol ester content was increased more than that of triglyceride by incubation with VLDL in the presence of carboxyl esterase and THPF. These results indicated that carboxyl esterase may be involved in the VLDL catabolism in the arterial wall by co-operation with THPF.

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Serum HDL₂- and HDL₃-cholesterol (HDL₂-C, HDL₃-C) levels were determined in healthy children and adults by polyacrylamide gradient gel electrophoresis.
Adult females have higher HDL-C levels than adult males (p<0.01). This difference seemed to be due to higher HDL₂-C levels. No significant differences existed in any subfraction in children.

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Apolipoprotein A-I, A-II, B, C-II and E were determined by a single radial immunodiffusion method on fasting blood samples from 554 (70%) of 786 in habitants aged above 35 in Wara Village in Gifu Prefecture.
All five apolipoproteins showed higher values in males than in females until forty, but in those aged above fifty, A-I and B marked higher values in females than in males while differences were hardly seen among them for A-II, C-II and E. Regardless of their ages, TG and C-II significantly showed positive correlation both in males and females. Also in those aged above forty, a significant correlation was seen between total cholesterol and B, and between HDL-cholesterol and A-I, respectively.

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Ninety male rats were prepared for studying the effect of X-ray radiation on vascular wall cells with the aging.
They were divided into three groups as young (80g), adult (280g) and old (350g). Two cotton pellets were inserted into the rat's shoulders to cause the increase of ³H-thymidine incorporated cells in the aorta and heart.
X-ray radiation had remarkably inhibited ³H-thymidine incorporated cells in the aorta and heart especially in the young rat group.
In the three layers of aorta walls a remarkable decrease of ³H-thymidine was noted in the media in the young group after the X-ray radiation.

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Wistar rats were placed in an orthostatic state 3 hours every day for 11 months.
Arteriosclerotic intimal thickening of the small coronary arteries accompanied by small infarctions of the myocardium were detected. Neither necrotic nor PN-like lesions were seen.
Blood pressure increased gradually and showed levels of 180mmHg at the end of the experiment.
Further studies are necessary to determine the cause of intimal thickening of coronary arteries, that is, whether it derives from orthostatic circulation or from a hypertensive condition following orthostatic experiment.

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We observed the pathophysiological state of a colony of Wistar rats with a hereditary defect in vitamin C (L-ascorbic acid)-synthesizing ability. This rat lacks L-gulonolactone oxidase in liver or adrenal gland which catalyzes the last step of Lascorbic acid biosynthesis, and this strain was named the osteogenic disorder rat (OD rat). It was found by Makino et al in 1973, and the strain controlled by a single autosomal recessive gene with the gene symbol od was established from Wistar rats.
We found that OD rat has some atherogenic risk factors, that is, hypo-HDL-cholesterolemia, hyper platelet aggregability and malmetabolism of aortic collagen. Serum HDL-cholesterol level was able to increase by the supplementation of L-ascorbic acid.
In these studies, we could not find the atherosclerosis in this mutant strain. But we may detect the process of atherosclerosis if its life span can be elongated.

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Recently coronary artery spasm has become able to be induced experimentally. It has been reported that spasm was more easily induced when sclerotic changes pre-existed in the arterial walls. This study was attempted to study whether coronary spasm might accelerate a permeability in the coronary arteries, and proceed to further sclerotic changes.
The experimental group of dogs had been pretreated with denuding of endothelium of the left anterior descending arteries by a coil tip through coronary catheter with simultaneous ECG monitoring. Then, methacholine was injected intramuscularly, 1 time, 5 times, 10 times and 20 times, for 1 day to 27 days of total experimental period. The methacholine injection at the last time was under ECG monitoring, and when ECG showed ischemic changes such as ST elevation or depression, coronarycinearteriography was performed. Among 11 dogs, five showed spasm in non-denuded segments, usual further distal to denuded areas, of the coronary arteries. Evans blue was injected continuously into the pulmonary arteries for about 3 minutes. Ferritin also was injected at the same time, as a tracer for electron microscopical studies. After the coronary arteries were resected, they were cut through the long axis, and gross stain by Evans blue was observed on the intimal surface, then cut crossly at every 5mm width, each tissue block was numbered, and the odd numbers were made into microscopic sections. In every spastic segment, gross stain by Evans blue was observed and abnormal changes were found in intima and internal elastic layer, such as slight thickening of the intima and increased intimal cells, and fraying and widened fenestration of the inner elastic layer. Electron microscopical studies showed ferritin particles in inter-cellular spaces of intima and just beneath the internal lamela of media. Fat stain showed fine droplets besides the inner elastic layer.
These obtained results by the experiment may lead to a conclusion that repeated spasm makes abnormal changes of the intima and inner elastic layer, and vascular permeability increased. This fact suggests that spasm may be one of the risk factors of coronary sclerosis.

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An increase in Ca-content of erythrocytes can induce the characteristic changes, such as reduced osmotic fragility and increased rigidity of the membrane. These phenomena are induced by the binding of the intracellular Ca to the cytoskeleton proteins (contractile proteins) distributed in the erythrocyte membrane.
To investigate the Ca-sensitivity of the erythrocyte membrane, the changes of the osmotic fragility of erythrocytes by Ca-loading were observed. Samples from essential hypertension (WHO I and II) and spontaneously hypertensive rats (SHR, Okamoto & Aoki) were examined in comparison with age-matched normotensive subjects and Wistar Kyoto rats (WKY), respectively. Treatment of washed erythrocytes with Ca-ionophore (A 23187) and Ca in the bathing medium caused the reduction of the osmotic fragility dose-dependently on Ca-concentration. The degree in alteration of the osmotic fragility by Ca-loading was significantly greater in hypertension than normotensive controls. However, in the presence of Ca-antagonists (verapamil, diltiazem) or calmodulin antagonist (trifluoperazine) in the medium, the reduction of the osmotic fragility by Ca-loading was inhibited, and the differences between the hypertensives and the controls were abolished by Ca-antagonists and a calmodulin antagonist.
These results suggest that greater changes of the osmotic fragility of erythrocytes by Ca-loading in hypertension might be due to a genetic abnormality of the Ca-handling of the cell membranes, and it would be corrected by Ca-inhibitors.

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Effects of the administration of dextran sulfate at a dose of 5mg/kg body weight on serum lipoproteins and apolipoproteins were investigated in normo- and hyperlipidemic subjects.
At 30min after dextran injection, serum free fatty acid and glycerol concentrations were elevated markedly, but declined already after 60min. On the other hand, serum triglyceride concentrations decreased to about 65per cent of the control level at 30min and kept the low level at 60min. These net changes were much larger in hypertriglyceridemic subjects than in normolipidemics. HDL cholesterol level was gradually elevated by 60min, which was due to the elevation of HDL₂ cholesterol. HDL₃ cholesterol was not influenced by dextran administration.
Serum VLDL concentration decreased to 41per cent of the pretreatment level. Although IDL, LDL and HDL concentrations increased after dextran injection, the changes of IDL and LDL were not significant statistically, because the changes of individuals were markedly variable. Only HDL showed significant increase.
Every component of VLDL was reduced by the treatment, but the decrease of apo C-II and C-III proteins in VLDL were more prominent than that of apo B. Therefore, VLDL became rich in apo B and poor in apo C-II and C-III by the treatment. By contrast, apo C-II and C-III increased in HDL fraction, though no consistant changes were observed in IDL fraction.
VLDL, VLDL apo C-II, C-III and E protein contents were well correlated to the changes of HDL apo C-II, C-III and E as well as those in VLDL and their apolipoproteins. VLDL triglyceride and its reduction were also correlated to triglyceride concentrations and these changes in other lipoprotein fractions and the sums of their changes.

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In order to study the effect of melinamide (ML) on plasma lipids, lipoproteins and apolipoproteins, 17 primary hypercholesterolemia (59±13 years old) were subjected to treatment.
Treatment by ML 1, 500mg/day for 8 weeks (During: D) significantly decreased plasma total cholesterol (P-Ch) concentration from 323mg/dl to 288 (p<0.001). Additional therapy by ML 2, 250mg/day for more than 8 weeks (After: A) further decreased P-Ch concentration to 264mg/dl, significantly (p<0.001). HDL-Ch level was also decreased significantly (p<0.005) from 63±16mg/dl (before treatment: Before: B) to 54±13mg/dl (D) and 51±13 (A). Cholesterol concentrations in very low and low density lipoprotein were significantly decreased from 260±32mg/dl (A) to 234±25mg/dl (D) and 213±33 (A).
Although plasma apo A-I level was not significantly changed between B: 136±19mg/dl and D: 134±20, after treatment by ML 2, 250mg/day apo A-I was significantly increased to 149±17mg/dl. Apo B concentration was decreased from 125±17mg/dl (B) to 118±8 (D) and 119±12, although this change was not significant. Apo E concentration was significantly decreased (B: 5.1±0.4mg/dl and D: 5.0±0.7 to A: 4.6±0.8mg/dl). Plasma apo A-II, C-II and C-III levels were not changed significantly.
In HDL composition, apo A-I/apo A-II ratio was increased (4.15±0.41 (B) and 4.01±0.33 (D) to 4.59±0.51 (A)) and change between D and A was significant. Apo A-I/HDL-Ch ratio was significantly increased from 2.2±0.4 (B) to 2.6±0.5 (D) and after treatment by ML 2, 250mg/day this ratio increased to 2.9±0.6, significantly.
This study revealed that melinamide is efficient drug for hyper cholesterolemia and that it changed HDL composition to more apo A-I rich particles.

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We studied the effects of Melinamide on serum levels of total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C) and apoproteins (A-I, A-II and B).
Both of serum TC and TG significantly (p<0.001 and p<0.05, respectively) decreased after oral administration of this agent for 6 months. Serum HDL-C significantly (p<0.05) increased, following to the decrease of serum TG. There were no significances in the changes of serum apoprotein levels after oral administration of this agent.
These results indicated that Melinamide was useful in the treatment of not only hypercholesterolemia but also hypertriglyceridemia. The inhibitory effect of this agent on the absorption of dietary cholesterol would induce the decrease of serum TC. However, it was thought that serum apoprotein metabolism was not influenced by this agent.

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The clinical non-invasive methods for the differential and quantitative diagnosis of arteriosclerosis were developed in order to predict and to prevent the arteriosclerotic vascular diseases. The purpose of the study is to reveal the significance of the hyperlipoproteinemia in the progression of atherosclerosis in Japanese. The subjects of the study were 194 out- and in-patients, of whom 68 normolipidemics, 41 type IIa, 54 type IIb, and 31 type IV or V hyperlipoproteinemic patients were involved. All of them were examined about the severity of atherosclerosis expressed by the wall thickening and stenosis index (S. I.) and the calcification index (C. I.) of the lower abdominal aorta gotten from the computed tomography after or before the enhancement. The mid-band lipoprotein concentration was calculated from the mid-band ratio of the electrophoresis pattern of lipoproteins using 30% polyacrylamide gel and VLDL and LDL levels. Atherogenic index of lipids was obtained by the formula of (total cholesterol-HDL cholesterol)/HDL cholesterol.
The mid-band lipoprotein concentration was significantly correlated with atherogenic index. Both the mid-band lipoprotein and atherogenic index values were the highest in type IIb hyperlipoproteinemia and the lowest in normolipidemics. The mid-band lipoprotein also correlated with total cholesterol, triglyceride and HDL cholesterol, significantly. No significant correlation was observed between the mid-band lipoprotein and S. I., but atherogenic index inclined to be correlated with S. I. in fifth decade. Patients with type IIb and IV or V hyperlipoproteinemia tend to have more progression in S. I. than in C. I., on the contrary, type IIa inclined to C. I. Patients with hypertension and/or diabetes mellitus in addition to various types of hyperlipoproteinemia got higher value of S. I. and C. I. and turn to the direction of C. I. The mid-band lipoprotein in patients with effort type of coronary heart disease was 207.5mg/dl and higher than those in patients with vasospasm type of coronary heart disease. In progression of atherosclerosis, great differences were showed between male and female and by age. Analysis in larger populations should be required to avoid the influence of sex and age differences.

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Cholesterol oleate liquid crystals were prepared in vitro as a model of lipid droplets accumulated in the atheromatous aorta. The hydrolysis of cholesterol oleate crystals by lysosomal acid lipase was examined in the presence and absence of various phospholipids. Phosphatidylserine markedly stimulated the hydrolysis of cholesterol oleate liquid crystals (20 times the basal value); it increased the V_max value about 15 times and decreased the K_m value to 1/20 times the basal value. The polar head group and the acyl chains of phosphatidylserine were required for its stimulation of hydrolysis.

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Effects of Intralipid on the increase of esterified cholesterol (EC) in each lipoprotein fraction in the fresh fasting plasma (FP) or triglyceride lipaserich plasma (PHP) from normal, and type V hyperlipoproteinemic subjects were investigated. FP or PHP was mixed with ¹⁴C-FC-albumin solution and was preincubated at 4°C for two hours. The mixture was incubated with Intralipid or with 0.15M NaCl solution. Incubation mixtures were collected at 0, 120 and 240 minutes. Chylomicron, VLDL, IDL, LDL and HDL were fractioned ultra-centrifugally at 4°C. Lipids were extracted with chloroform-methanol (2:1) and EC and FC were separated by TLC. The radioactivities of them were measured with a liquid scintillation counter. In the normal FP, Intralipid significantly increased ¹⁴C-EC in chylomicron, and VLDL fractions. However, Intralipid reduced cholesterol esterification, and this was solely due to the complete suppression of ¹⁴C-EC increase in HDL in normal PHP. The specific activities of EC in HDL were significantly lower than those in LDL in normal PHP with Intralipid. In FP of type V hyperlipoproteinemia, the increase of ¹⁴C-EC was more in chylomicron and VLDL fractions and less in LDL and HDL fractions than that in normal FP. The effect of Intralipid addition was very small in FP of type V hyperlipoproteinemia. In PHP of type V hyperlipoproteinemia, the results were very similar to those in normal FP with Intralipid. No increase of ¹⁴C-EC was observed in HDL. The effect of Intralipid addision was also small as in FP. Therefore, we concluded as following. Intralipid worked like chylomicron. There were two pathways for cholesterol esterification in the plasma. One was active in HDL and was blocked almost completely by Intralipid or chylomicron during the lipolysis. The other was active in other lipoproteins and was not inhibited by Intralipid or chylomicron during lipolysis.

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Age-related changes in the lipid and lipoprotein metabolisms of Fisher-344 rats were investigated in three groups of rats at 6 (N=9), 12 (N=8) and 30 (N=8) months of age. Plasma triglycerides and cholesterols were determined using enzymatic methods. Serum lipoproteins were analyzed by the High Performance Liquid Chromatography by method of Hara et al.
Plasma triglyceride concentration increased significantly with age. Serum lipoproteins, however, did not increase significantly. Measurement of glycerol by High Performance Liquid Chromatography revealed that plasma glycerol concentration increased with age. Plasma cholesterol concentration increased significantly with age. HDL-cholesterol concentration increased significantly with age, while VLDL and LDL did not change. These results suggested that increased amount of plasma triglycerides results from false-positive increase of glycerol components and increased amount of cholesterol with age results from the increase of HDL-cholesterol.

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Assay system of LDL receptor in rat hepatocytes using human LDL has been developed.
¹²⁵I-LDL association to hepatocytes and its degradation were measured at 37°C for 5 hours. About 50% of the associated ¹²⁵I-LDL was degraded both in isolated as well as cultured hepatocytes. Binding accounted for 50% of the association.
In cultured rat hepatocytes, cell association and degradation of ¹²⁵I-LDL decreased to 50% and 40% compared with isolated rat hepatocytes respectively, and binding also decreased to 50%. From the result of scatchard analysis, LDL receptor numbers decreased in cultured hepatocytes to 50% compared with that in isolated hepatocytes, while affinity did not change.

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Serum cholesterol and Apo B significantly decreased during 2 hours of insulin infusion in normal and diabetic subjects, but not in heterozygous FH who only have 50% of LDL receptor activity. Cholesterol in VLDL+IDL & LDL fractions also decreased in diabetic subjects, but no significant change was observed in FH. The result indicate that insulin has an early stimulating effect for LDL pathway.

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We examined low density lipoprotein (LDL) receptor activities of 13 heterozygous patients with familial hypercholesterolemia (FH) using peripheral lymphocytes.
The receptor activities were measured as the degradation of ¹²⁵I-labeled LDL in lymphocytes which had been cultured for three days in 10% lipoprotein-deficient serum. After incubation, 10μg/ml of ¹²⁵I-LDL were added to the medium in the presence or absence of 250μg/ml of nonlabeled LDL and then the lymphocytes were incubated for five hours further. The radioactivity of the trichloroacetic acid-soluble fraction of the medium was measured.
As a result, LDL receptor activities of FH heterozygotes were revealed to correlate significantly and negatively with serum LDL-cholesterol concentration. If we combined them with those of two FH homozygotes and 18 hyperlipidemic patients other than FH and normolipidemic persons, the latter of which we reported previously (J. Jpn. Atheroscler. Soc. 12: 303-308, 1984), not only the grade of the negative correlation between LDL receptor activities and LDL-cholesterol became greater, but LDL receptor activities also correlated negatively with total cholesterol (TC) (p<0.01). Extrapolation from these data suggested that LDL receptor activity would disappear when TC concentration was higher than 718mg/dl. The value estimated agreed well with the clinical findings of FH.
Then, probucol (750mg a day) was administered to eight heterozygous patients with FH for four to six months, by which TC concentration decreased significantly from 319.6±36.7 (mg/dl) to 282.3±17.4 (mg/dl). The result suggests that probucol is effective in the treatment of FH. The levels of LDL receptor activities in the pretreatment state was not correlated to the effectiveness of probucol treatment in each case.
LDL receptor activities were also measured after probucol administration and were compared with those of the pretreatment state. There were a great variations of change of LDL receptor activity among the eight patients, and no definite trend of the activity could be found. But, it was suggested that the percentage change of TC concentration was correlated negatively with the difference in LDL receptor activity before and after probucol administration, that is, the more the TC concentration decreased, the more the LDL receptor activity increased.
These results may suggest that the main action of probucol is not on LDL receptor and probucol indirectly influences LDL receptor activity by the change in serum cholesterol concentration.

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