This study was designed to clarify the role of nutrition and physical exercise for preventing hyperlipidemia and hypertension.
Sprague-Dawley strain rats were fed on a hypercholesterolemic diet containing 0.5% cholesterol and 0.25% Na cholate. Safflower oil suppressed hypercholesterolemia induced by dietary cholesterol when it was administered as the dietary source of fat. In contrast, administration of lard did not affect such hyperlipidemic state. Serum HDL cholesterol level had a tendency to increase in safflower oil group.
Voluntary or forced exercise was loaded on the hypercholesterolemic rats. Both exercises significantly decreased serum triglyceride. Serum total cholesterol level was lowered by voluntary exercise rather than forced one. A reverse correlation was observed between serum total cholesterol and the running distance in voluntary exercise group when the animals were fed sucrose as carbohydrate source. The correlation was lower when starch or fructose was used in place of sucrose.
Experiments were conducted to investigate the hypotensive principle (s) in Shii-ta-ke (Lentinus edodes) fruit bodies as well as in their mycelium on the blood pressure of spontaneously hypertensive rats (SHR). The extract of both fruit bodies and mycelia decreased the blood pressure of SHR. Safflower oil, Soya lecithin and highly purified eicosapentaenoic acid were tested in the same manner, and were found to exhibit obvious hypotensive activity.
Healthy adult volunteers were supplied for 7 days several kinds of lipid-containing food. Combined administration of butter and eggs remarkably increased serum total cholesterol after 7 days, but single administration of each one did not. Blending oil of rice bran and safflower oil, in contrast, significantly decreased serum total cholesterol and increased HDL cholesterol.
These results suggested a close relation between nutrition and exercise in the prevention of hyperlipidemia and hypertension.

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1) High protein diet intake in the younger age lowered hypertensive complications resulted from arterionecrosis even though relatively low protein diet was given in the older age, and the development of such complications was still preventable by high protein diet intake in spite of the low protein intake in the younger age.
2) Marked preventive effects of high protein diets on the development of fat deposition in cerebral and mesenteric arteries was substantiated. Especially, such a beneficial effect was remarkable when rats were given a fish protein rich diet containing richly sulfur amino acids with both BP and serum Tch lowering effects.
3) Vascular SMC from SHRSP incorporated more lipid when hyperlipidemic serum was added in the culture medium than SMC from WKY and this intracellular lipid deposition showed a faster regression in the medium with FCS than without.

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To confirm the relationships between vessel injuries and sugar intake, the following experiments were carried out respectively.
Experiment 1
Natural history of SHRsp and SHR in the administration of various feeds.
Experiment 2
The observations of vessel changes by light microscope and scanning electron microscope in various feeds.
The incidence of cerebral bleeding was higher in the combination of sugar feed and hypertension than those in other combination. And also the vessel changes of aorta, brain heart and kidney in the same condition were prominent.

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The contributory effect of amino acids supplementation on the development of hyperlipidemia was investigated in uremic patients on maintenance hemodialysis. Hemodialysis treatment lost large amounts of carnitine and other amino acids from blood into the dialysate fluid, resulting in the reduction in serum concentration of Carnitine. After the treatments were repeated for more than 12 months, serum carnitine concentration reduced markedly with significant increases in serum levels of triglyceride and VLDL. In contrast, in patients who had been supplemented with commercial amino acids solution at the end of each treatment, serum concentrations of carnitine, triglyceride and VLDL were within normal ranges even after repeated hemodialysis treatments, because supplementation with the solution corrected serum amino acid composition with an increased synthesis of carnitine. In these patients post-heparin lipoprotein lipase activity (PHLA) and apoprotein C-II level were much higher than those in patients who had not been supplemented with the solution. Carnitine administration also reduced serum concentrations of triglyceride, VLDL and apoprotein C-II and PHLA to or towards normal. The results suggest that carnitine depletion induced by hemodialysis treatments causes hemodialysis hyperlipidemia in uremic patients and that supplementation with amino acids solution prevents carnitine depletion and improves hyperlipidemia.

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There is an apparent tendency in fat intake for Japanese which is gradually increasing in the last few decades, with concomitant increase in the incidence of the coronary heart disease.
The present study was conducted to investigate whether the alterations of fatty acids in the high-fat diet would change lipoprotein lipids, apoproteins and blood pressure of normal Japanese individuals.
Thirty eight healthy volunteers were subjected to receive either 40gr of margarine rich in linoleic acid or the same amount of butter for 4 weeks in cross-over fashions.
Compared with the results in butter period, total cholesterol, LDL-cholesterol and HDL-cholesterol decreased in the margarine period.
No significant differences were observed in apolipoprotein A-I, A-II, B, C-II, C-III and E concentrations between two experimental periods.
Decrement of plasma total cholesterol was greater in the subjects showing the changes more than 5% in plasma cholesterol linoleate than in the subjects showing less changes.
Decrease in the systolic blood pressure in the margarine period seems more frequent in 3 weeks than in the butter period.
Also the subjects with lowered systolic blood pressure more than 5mmHg indicated the significant increase of the plasma cholesterol linoleate in the margarine period.
It is concluded from the present results that high polyunsaturated fat-rich diet would be more preferable to prevent from the elevation of the blood cholesterol and blood pressures.

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EPA has been known as an anti-thrombotic agent.
We investigated the mechanisms of its antithrombotic effect from epidemiological and subsequent laboratory studies.
Epidemiological study revealed the low incidence of cardiovascular disorders among the residents in a fishing village who have reduced platelet aggregability and whole blood viscosity than those in a farming village.
Futhermore we prepared highly purified EPA etyl ester (EPA-E) and administered them to 64 patients with atherosclerosis and hyperlipidemia.
Sixteen weeks administration of EPA-E resulted in prolonged bleeding time, reduction of platelet aggregability and retention, improvement of rheological properties and reduction of serum lipids among the patients on this clinical trials without any side effect.
These studies suggest that hemostatic function could be manipulated with dietary EPA and that the ingestion of EPA rich diet could be beneficial in the treatment and the prevention of atherosclerotic disorders.

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We set out to establish whether dietary sodium restriction is feasible and effective in the treatment of essential hypertension on general practice.
Fifty-six patients with mild to moderate essential hypertension in outpatient clinic, whose average blood pressure was 169±20/103±8.9mmHg and average relative body-weight was 1.25 (range 0.93-1.95) were allocated to 3 groups by the reduction of urinary sodium-excretion and/or bodyweight after the dietary instruction for hypertension. These patients on non-medication for hypertension were randomly instructed on how to reduce sodium intake or energy intake by metabolic ward dietician. He checked the patients about the compliance of dietary instruction using a questionnaire, and a change of urinary sodium-excretion and body weight at every 6 weeks for 12 weeks.
50% of the patients, whose 24-hour urinary sodium-excretion were decreased without a decrease in body-weight, were effective on blood pressure. In 7 of 8 effective patients, 24-hour urinary NaCl-excretion before instruction were 12g or more and the magnitude of a reduction of urinary NaCl excretion was more than 3g/day.
On the other hand, only 30% of patients whose body-weight decreased without a decrease in sodium-excretion, were effective on systolic pressure. But urinary sodium-excretion in this group increased, especially that in non-effective patients was higher than in effective patients and plasma aldosterone in non-effective patients significantly decreased from 10.0±2.6 to 8.5±3.4ng/dl (p<0.05).
68.8% of patients with decrease in both sodiumexcretion in urine and body-weight were effective, especially frequency of the patients with decrease in diastric pressure was significantly high (χ²=5.11, p<0.05). Though plasma aldosterone level did not change after decrease in both sodium excretion and body-weight, plasma insulin level significantly decreased in only effective patients (p<0.05).
In 26 of all patient with decrease in body weight, fasting plasma insulin level were 7μU/ml or more before dietary instruction. 12 of 15 patients, whose plasma insulin level decreased in 2μU/ml or more after dieary instruction, were decreased in blood pressure.
Conclusion: The dietary salt restriction which were reducted by 3g/day or more may be effective to hypertension when hypertensive patients have over 12g/24-hour urinary NaCl excretion. But it may be necessary in an effectiveness of diastric hypertension to make body weight reduction and the effect of weight-reduction on hypertension appeared when relatively high concentration of plasma insulin make decrease.

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An improved method was devised to estimate separately HDL₂ and HDL₃ by emproying a polyacrylamide-gel electrophoresis (PAGE).
To serum or plasma was added a equal volume of the goat antiserum against human β-lipoprotein followed by centrifugation, and the supernatant which contains α-lipoprotein was prestained with Sudan-Black B. The stained material was subjected to PAGE with 7.75% gel for about 60 minutes at 4mA/one column. Our modification has two significant advantages; the method enables better separation of the two subfractions and it prevents the loss of the Sudan-Black B staining. It was noticed that a considerable amount of HDL was lost when Sudan-Black was added directly to serum (as in the original method by Miyahara) for some unknown reason, but when the staining was performed with the pre-separated α-fraction, the loss was much smaller. Also the use of 7.75% gel gave rise to a more distinct separation of subfractions as compared with those obtained with 6.75% gel. The application of the newly devised method provided the following findings. 1) The ratio of HDL₂/HDL₃ in women was twice as much that in men. Difference in ratio between young and old, however, was not significantly. 2) Cases showing a high HDL₂/HDL₃ ratio were found only in women. Higher HDL-C and lower TG were demonstrated in the cases with high HDL₂/HDL₃ ratio as compared with those in the low ratio group. 3) Convention of HDL₃ to HDL₂ was attempted by incubating the serum for 24hrs. at 37°C and it was found that the mobilities of HDL₃ and HDL₂ were decreased to 80 and 85%, respectively, as compared to their mobilities in the untreated serum. When the serum was added with PCMPS, a inhibitor for LCAT, before the incubation, the decrease of the mobilities was smaller; the extent of decrease was less than 10%.

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We have determined alpha-tocopherol (TOC) levels of red blood cell (RBC), serum and high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) method in 10 uremics and in 10 age-matched normal controls. All subjects were male.
TOC levels of RBC, serum and HDL were significantly reduced in uremics, particularly hemodialysis patients, than in controls.
In the control group, there was a positive correlation of RBC to HDL and HDL/serum-HDL (non HDL) ratio while a negative relationship between RBC and non HDL was observed. On the other hand, the uremic group had only a negative relationship between RBC and HDL.
The results in this study demonstrated that low RBC-TOC levels in uremics may be due to an impairment of TOC transfer from HDL to RBC.

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Though, some studies on the level of vitamin E in blood cells have been reported since 1978, its clinical significance has been still equivocal.
We reported here the levels of vitamin E in serum and in blood cells of vitamin E deficient and supplemented rats. Serum vitamin E were 0.74±0.33mg/ml in control, 0.57±0.05 in deficient and 1.30±0.20 in supplemented. Platelet vitamin E were 13.11mg/10¹⁰ cells, 0.00 and 25.46 in each and RBC vitamin E were 1.46±0.07mg/10¹⁰ cells, 0.00 and 5.05±0.12 in each.
The maximum aggregation rate of ADP-induced platelet was highly accelrated in vitamin E deficient group, while significantly reduced in supplemented group. Concerning about the fatty acid composition in platelet, only linoleic acid was markedly reduced in vitamin E defficient platelet.
These results reminded us that the dietary vitamin E affected more strongly on the vitamin E level of blood cells than that of serum and it contributed the enhancement of platelet aggregability which is widely recognized as the promoter of atherosclerotic process.

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Diabetes mellitus is one of the atherogenic diseases. We should improve not only the derangement of glucose metabolism but also the hypercholesterolemia in diabetics. Clinofibrate (Lipoclin^R) is clinically useful in hypercholesterolemia. In order to evaluate the effect of this drug on serum cholesterol level, we studied 106 hypercholesterolemic diabetics.
Clinofibrate (600mg/day) was orally administered to each patient. Their serum lipids were measured before and every 4 weeks for 24 weeks after administration of this drug.
The results were that (1) serum total cholesterol levels were significantly (p<0.001) decreased after Clinofibrate administration, (2) this decrease was observed not only in well controlled diabetics (fasting blood glucose, FBG<120mg/dl) but also in fairly controlled diabetics (FBG 120-179mg/dl) under stable condition, and (3) this decrease was found in the case with persistent proteinuria.
In conclusion, this drug is useful to decrease serum cholesterol level in hypercholesterolemic diabetics.

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To evaluate the additive hypolipidemic effect of a simultaneous administration of clinofibrate and melinamide, 11 patients with type II hyperlipoproteinemia in whom cholesterol levels were 240-300mg/dl and familial hypercholesterolemia was not included were selected. They were treated with clinofibrate in a dose of 600mg a day for 8 weeks, followed by a simultaneous administration of clinofibrate in the same dose and melinamide in a dose of 2, 250mg a day for another 8 weeks.
The serum level of total cholesterol was decreased by 10.2% after the 8 week treatment and by 15% after the 16 week treatment. The serum level of triglyceride was decreased by 15.6% and 29% after the 8 and 16 week treatments respectively. The serum levels of high density lipoprotein-, HDL₂-, and HDL₃-cholesterol remained unchanged. However, atherogenic index—(total cholesterol—HDL-cholesterol)/HDL-cholesterol—was decreased significantly. In addition, the serum level of apo A-I was increased and that of apo C-III was decreased significantly. No side effects were observed throughout the study.
These additive hypolipidemic effects were considered to justify the simultaneous administration of clinofibrate and melinamide when a single administration of either of these two drugs could not achieve a sufficient effect for preventing the progression of atherosclerotic vascular disease.

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Dilazep at daily dose of 300mg were given orally for 12 weeks to 83 hyperlipidemic patients with ischemic heart disease or diabetes mellitus visited to the designated hospitals and effects on their serum lipid, apolipoprotein A concentrations were investigated.
Although there were no definite effects of the drug on serum total cholesterol, triglyceride and phospholipid concentrations, HDL cholesterol and HDL/VLDL-LDL cholesterol ratio were elevated significantly. Since HDL₂ cholesterol increased by the treatment, HDL₂/HDL₃ cholesterol ratio was elevated. Apo A-I protein increased succesively after the change of HDL cholesterol, and apo A-II protein decreased inversely by Dilazep. Consequently, apo A-I/A-II ratio after the treatment became higher than that in the pretreatment period.
The effects of the drug on HDL cholesterol, apo A-I and A-II protein concentrations were more pronaunced in the patients with low HDL cholesterol below 35mg/100ml than in other subjects, but no consistent change of HDL₂/HDL₃ cholesterol ratio was found in such patients. The similar effects on serum lipids and lipoproteins were observed in type IIa, IIb and IV hyperlipidemia.

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The antiatherosclerotic effects of dilazep and/or aspirin were studied on experimental atheroselerosis which was induced by cholesterol loading in male New Zealand White rabbits. After 12 weeks of cholesterol loading and subcutaneous drug administration, aorta was removed to measure lipid deposition and intimal thickening. The former was measured by Xerographic method and the latter was done by histological technique. In control group, about 62% of aortic surface was covered by deposited lipid and intima was extremely thickened not only in aortic arch but also in thoracic aorta. Dilazep showed more suppressive effect than aspirin on lipid deposition and intimal thickening especially in thoracic aorta and these effects were strongly enhanced by the combination of two drugs. Dilazep and aspirin, however, showed no effect on hyperlipidemia. From these results it seems to be suggested that by the different mechanism from hypolipidemic agents e.g. clofibrate, both drugs showed antiatherosclerotic effects. Antiplatelet activity of aspirin and antiplatelet, fibrinolytic and membrane stabilizing activities of dilazep could be considered as mechanisms of antiatherosclerotic effects of both drugs.

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We reported the effect of MDS both on the reduction of serum lipids and the prevention of coronary heart diseases in previous report, on which the administration of MDS 1, 800mg per day was clarified to be effective on the prevention of coronary heart diseases. Moreover, the improvement of serum lipid levels was observed in this trial. Namely, the significant reduction of serum total cholesterol (TC) was observed in type IIa of MDS group, while the significant reduction of serum triglyceride (TG) was observed in type IIb of MDS group. The significant increment of serum HDL-cholesterol was also observed in type IIa and type IIb of MDS group. Atherosclerotic index (TC-HDL-chol/HDL-chol) improved in type IIa and type IIb of MDS group. This study showed that the improvement of serum lipid levels with long-term administration of MDS (1, 800mg/day) can prevent the accidents of the ischemic coronary heart disease primarily and significantly.

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Though there were several reports on antiatheromatous effect of glycosaminoglycans (GAG), the biological role of GAG remains still controversial. In order to make clear whether GAG has an antiatheromatous effect or not, male Japanese white rabbits were housed for 10 weeks with three different diet as follow; (1) group [A], fed by standard diet, (2) group [B], fed by 1% cholesterol diet, (3) group [C], fed by 1% cholesterol diet with 10mg of C-6-S. C-6-S was administered with the intraperitoneal injection, while saline were given other groups in a same manner. (Results); Macroscopically, atherosclerotic lesions of aortic surface of group [C] was significantly lesser than those of group [B]. Cholesterol contents of aortic intimamedia of group [C] (1.6±0.18mg/100mg.d.w.) was significantly lower than that of group [B] (2.9±0.33mg/100mg.d.w.) (p<0.01). Uronic acid contents of aortic intima-media in group (A), [B] and [C] were 343±16.3, 370±11.2 and 305±17.4μg/100mg.d.w., respectively and there were statistically significant differences between group [B] and [C] (p<0.01). The %HS of group [C] showed lower than that of group [B], while there were no significant differences in %DS and %C-4/6-S between both groups. From these results, it might be suggested that C-6-S suppress the cholesterol deposition in rabbit aorta on 1% cholesterol diet and also suppress the accumulation of GAG which probably reflect the progression of atherosclerosis.

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The migration of vascular endothelial and smooth muscle cells has a very important role in the initiation and progression of atherosclerosis. And we devised a method to estimate the repair of vascular endothelial monolayer in culture. We can observe the migratory activity isolated from the proliferative activity of the cells by adding an inhibitor of DNA synthesis, hydroxyurea, in the medium. This method is technically easy and is also applicable to other cells, such as smooth muscle cells in culture.
In this paper, we reported the method in detail and preliminarily the effect of diltiazem on vascular endothelial cells and smooth muscle cells.

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We studied the effects of the calcium antagonist diltiazem on the development of atherosclerosis in rabbits pathologically and biochemically. Twenty male Japanese white rabbits weighing about 2.0kg were divided randomly into three groups. Group 1 (n=7) rabbits were fed a standard diet plus saline. Group 2 (n=8) received a 1% cholesterol diet plus saline. Group 3 (n=5) received a 1% cholesterol diet plus 50mg of diltiazem. Saline and diltiazem were administrated by intraperitoneal injection. At the 10 week feeding the rabbits were sacrificed. The plasma total cholesterol level in group 3 was 757.7±158.6mg/dl (mean±SE), being reduced statistically compared with group 2 (1, 491.8±231.6mg/dl, p<0.05). Atheromatous aortic lesion covered 26.7±6.1% (mean±SE) of intimal surface in group 2 and 0.7±0.3% in group 3. Pathologically atherosclerotic lesion in group 3 was suppressed compared with group 2 and was almost the same as group 1. Mean values ±SE for the aortic cholesterol content and calcium concentration were 29.4±9.2mg/g.d.w. and 3.5±0.3mg/g.d.w. in group 2. Corresponding values in group 3 were statistically different, being; 8.8±3.8mg/g.d.w. and 1.6±0.1mg/g.d.w. We concluded that diltiazem suppressed the experimental atherosclerosis.

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We studied the effect of nifedipine, a calcium antagonist, on atherosclerosis of Watanabe heritable hyperlipidemic rabbit (WHHL-R). WHHL-R lacks in LDL receptor and is a unique model of human familial hypercholesterolemia. Rabbits were fed standard rabbit chow 120g daily, and were divided into two groups so that there were no significant differences in weights and serum cholesterol levels. Experimental group was given orally 20mg of nifedipine twice a day (nifedipine group), and control group was given placebo in the same way (placebo group).
Rabbits were sacrificed at the end of 20 weeks of treatment. Body weights and serum cholesterol levels were not different between nifedipine and placebo group. The percentage of intimal surface area covered by atherosclerotic lesions (% AS) was 33.4±14.1% in nifedipine group (n=5), 27.0±11.7% in placebo group (n=6) (n.s.). Aortic cholesterol and calcium contents showed similar tendency to the results of %AS.
Although many reports suggest that calcium antagonists suppress atherosclerosis in cholesterolfed rabbits, we concluded atherosclerosis was not suppressed by nifedipine in WHHL-R. This gives important suggestions to the mechanisms of antiatherosclerotic effect of nifedipine.

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In a series of 18 patients with essential hypertension, nicardipine HCl (60mg/day) was administered daily for. 32 weeks in A group (10) while propranolol HCl (30mg/day) was administered for 16 weeks and replaced by nicardipine HCl in B group (8), with the following results:
1. Mean blood pressure was significantly lowered in both A and B groups. In B group, drug replacement produced a further significant fall.
2. TC and AT significantly fell in A group, while they showed no significant change during propranolol treatment and significantly decreased after drug replacement in B group. Apo B underwent the same pattern of change.
3. HDL-C and LCAT significantly rose in A group, while being significantly elevated only after drug replacement in B group. Apo A-I was insignificantly elevated in A group and in the latter treatment in B group.
4. TX B₂ was significantly lowered at 32 weeks after the initiation of treatment in A group and at 32 weeks after that in B group, while 6-keto-PGF_1α showed no significant change.
From the above findings indicating that nicardipine HCl induced a fall in TC and a rise in HDL-C besides comparing favorably with propranolol HCl in producing a significant improvement, nicardipine HCl was considered an interesting drug in that it could improve the risk factor arteriosclerosis in the treatment of hypertension with a coronary risk factor.

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Pantethine is known to have hypolipidemic effect and to increase high density lipoprotein (HDL)-cholesterol. Effect of pantethine on serum lipids and apolipoproteins of 29 hyperlipidemics (12 type IIa, 13 type IIb, 4 type IV) were tested. The drug (600mg per day) was administered for 12 weeks after 2 to 4 weeks of diet therapy. There were “high responders” who showed more than 10% decrease in serum total cholesterol. The high responders were mainly found in type ha hyperlipidemics. There was no difference in sex, age, or serum total cholesterol levels between high responders and low responders (less than 10 decrease of total cholesterol). There was simultaneous decrease in total cholesterol and apo B levels, indicating little change had occurred in composition of lipoprotein. It was suggested that pantethine decrease very low density lipoprotein (VLDL) formation in liver and that this drug has little effect on catabolism of lipoprotein in peripheral tissues and catabolism of remnant lipoproteins in liver.

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The effect of Pantethine on serum lipids and apolipoproteins in non-insulin-dependent-diabetes was studied. Twenty patients under the stable conditions received Pantethine with a dose 600mg/day for six months or more. Levels of serum total cholesterol, triglyceride, HDL-cholesterol and apolipoproteins (Apo A-I, A-II, B, C-II, C-III and E) were measured every three months during the treatment.
The following results were obtained:
1) Serum total cholesterol decreased significantly after three months and six months of administration of Pantethine. Triglyceride tended to decrease after three months of administration of Pantethine.
2) HDL-cholesterol remains unchanged before and after the administration of Pantethine.
3) Apo A-I and Apo A-II decreased significantly after six months of administration of Pantethine.
4) Apo B decreased significantly after three months of administration of Pantethine.
5) Apo C-II and Apo C-III tended to decrease after three months of administration of Pantethine.
6) Apo E tended to decrease in the hypercholesterolemia group and the hypertriglyceridemia group after the administration of Pantethine.
7) Hemoglobin Al tended to decrease after the administration of Pantethine.
These results suggest that the administration of Pantethine with diabetic treatment would improve the abnormality of serum lipids and apolipoprotein levels in type II diabetics.

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β sitosterol is an agent clinically used for reducing wide-spread cholesterol. The effect of β sitosterol on dissolution of cholesterol gallstone, specially biliary lipids, serum lipids, serum apolipoproteins and apolipoproteins in lymph chylomicrons was examined in humans and rats.* Administration of β sitosterol produced the following results.
1) In humans, serum total cholesterol excluding high density lipoprotein cholesterol tended to decrease. Increment of apolipoprotein A-I was statistically significant (p<0.01).
2) The lithogenic index was statistically significantly (p<0.01) reduced in humans.
3) Decrement of serum cholesterol was statistically significant (p<0.05) in rats given cholesterol chow.
4) The output of biliary cholesterol, but not the pool size of total bile acids, was diminished significantly (p<0.05) in rats.
5) The ratio of apolipoprotein A-IV to A-I in the mesenteric lymph chylomicrons tended to decrease in rats given cholesterol chow.
The above results indicate that β sitosterol is an agent effective not only for reducing serum cholesterol but also for dissolving cholesterol gallstone. Further studies are needed to confirm the effect of β sitosterol on apolipoproteins.
* In humans, serum lipids, serum apolipoproteins and biliary lipids were measured. In rats, serum lipids, biliary lipids and apolipoproteins in the mesenteric lymph chylomicrons were measured.

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Arterial connective tissue appears to play an important role in the development of atherosclerotic plaques. The purpose of the present study is to examine the structural change in the connective tissue elements, especially elastin, of arterial wall during the lipid accumulation.
The nondelipidated portions of each lyophilized human arterial intima-media were fractionated to glycoprotein, collagen and elastin frs. Lipid was extracted from each fr. and its cholesterol content was determined. The defatted elastin fr, was hydrolyzed to the constituents amino acid, and amino acid composition and Lys originated crosslinking components were assayed on an automatic amino acid analyzer. The defatted collagen fr. and elastin fr, were solubilized then comparative hydrophobic interactions of collagen and elastin for hydrophobic ligands (octyl-sepharose CL-4B) was examined (expressed as a hydrophobic index).
The following results were obtained as the results:
1) Cholesterol content of the elastin fr. in the arterial wall of atherosclerotic regions or myocardial infarction was higher than that other disease (acute cardiac failure and chronic alcoholism). The data indicate that lipid deposition was seen mainly in the elastin fr.
2) Where more cholesterol deposit was observed, ratio of the cross-linking components (desmosine/Lys and isodesmosine/Lys) in the elastin fr. was reduced. The cholesterol content and the ratio were negatively correlated, and where more cholesterol deposition was observed, the amino acid in elastin fr, was changed.
3) The elastin fr. had stronger affinity for the hydrophobic property than collagen fr. It may be indicated that lipid deposition in the elastin fr, is due to its larger hydrophobic interaction. It may be assumed that the lipophylic proparty and reduced cross-linking components in arterial elastin fr, are due to altered composition of amino acids in elastin.

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A total of 336 subjects entered the study. Pulse wave velocity (PWV) was measured above 60 years (77.0±6.6 mean±SD, 145 men and 191 women). PWV gradually increased with age (r=0.29, p<0.01). In man mean ±SD PWV of normotensive 60-69 years, 70-79 years, and 80-89 years were 8.95±2.32m/sec (n=20), 10.27±2.11 m/sec (n=33) and 11.35±2.54m/sec (n=19) respectively. Hypertensive men showed higher but not significant PWV and their agewise increase was less than in normotensive men. Mean PWV±SD of normotensive women in sixties, seventies and eighties were 8.37±1.75 (n=16), 10.03±1.65 (n=38), 10.64±2.88 (n=28) m/sec respectively. Again those of hypertensive women were generally higher than of normotensive women but with less agewise increase. Total cholesterol and HDL cholesterol were not significantly correlated with PWV (r=0.06, -0.09), whereas LDL cholesterol was significantly correlated with PWV (r=0.11, p<0.05). HDL/LDL ratio showed significant negative correlation (r=-0.12, p<0.05), and low HDL/LDL ratio was significantly frequently observed in high PWV individual over. 15m/sec. Systolic blood pressure showed most positive significant correlation with PWV (r=0.30, p<0.01). Diastolic pressure was not significantly correlated. The results confirm the agewise increase of PWV and the accelerating influence of hypertension with less agewise effect and moderate correlation with LDL cholesterol.

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We have determined an aortic calcification index (CI) calculated from 8-10 slices of abdominal CT scan according to the method of Watanabe et al. and a pulse wave velocity (PWV), an indicator of aortic elasticity, by the method of Hasegawa et al. in 60 patients on maintenance hemodialysis (HD) and in 90 subjects without chronic renal failure including 16 normal controls (NHD). In addition, CI was compared with the degree of blood pressure, cardiomegaly, electrocardiogram (ECG) and PWV in HD.
CI in same generation, especially in erderly, was significantly higher in HD than in NHD.
There were no significant correlations of the presence and absence of CI to the frequency of hypertention, normotention and the ischemic changes of ECG whereas cardiomegaly tended to be highly related to the presence of CI.
CI in HD gradually increased in parallel to hemodialysis period. CI was highly correlated to PWV in NHD but not in HD.
The results obtained in the present study demonstrated that aortic calcification which could be an indicator of atherosclerosis was accelerated in HD as compared with that of NHD in same generation.

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Macrophage may play an important role in the atherogenic process because they appear to be precursors of some arterial wall foam cells. Recently, it has been reported that human peripheral monocytes in culture synthesize and secrete lipoprotein lipase (LPL). Consequently, it is possible that LPL secretion by human monocytes is related to atherogenesis. As shown below, guinea pig alveolar macrophages in culture synthesize and secrete LPL. Therefore we investigated the effect of lipoprotein on LPL activity of guinea pig alveolar macrophages.
Alveolar macrophages were cultured for 2 days with either RPMI-1640 medium containing 10% lipoprotein deficient serum (LPDS) or that medium with the addition of VLDL, LDL, and HDL respectively in the concentration of 300μg protein/ml.
When VLDL was added to LPDS after one day, LPL activity was significantly reduced to 48.3 within 2 days of exposure when compared with LPDS controls (p<0.02).
We believe there is a possible explanation for this phenomenon, LPL activity may be inhibited by apoprotein C-III of VLDL.

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The interaction of human skin fibroblasts with monocyte-derived macrophages in terms of accumulation of cholesterol ester in those cells were studied. Human intermediate density lipoprotein (IDL) was labeled with [3H]cholesteryl linoleate ([³H] IDL). Macrophages were incubated with [³H] IDL and this conditioned medium with macrophages was added to cultured human skin fibroblasts. The incorporation of the radioactivities into skin fibroblasts increased time-dependently compared to those of [³H]IDL conditioned without macrophages. By zonal ultracentrifugation of this conditioned medium 2 new peaks of radioactivities were observed. Among them, the radioactivitiy of Fraction III (density=1.15) was incorporated mostly into skin fibroblasts and increased dose-dependently cholesterol ester content of skin fibroblasts. It contained 51% of protein, 29% of cholesterol, 4% of triglyceride and 16% of phospholipid. These results indicated that IDL was incorporated and degraded by macrophages and that its metabolite was incorporated into skin fibroblasts and increased their cholesterol ester content.

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The role of myofibroblasts in angiogenesis was evaluated by morphological studies on microvessel formation in vitro. When microvessel fragments and myofibroblasts from rat epididymal fat pads were co-cultivated, myofibroblasts grew rapidly to confluence by 4 days of culture. Just 1-2 days later, endothelial cells started to sprout from the vessel fragments, forming a cellular cord network on the mono-, or multi-layered myofibroblasts. The lumina surrounded by endothelial cells with tight junctions were observed in the electron micrograph of cross-sectioned cellular cords on the 14th day of culture. On the other hand, when the microvessel fragments were cultured in the absence of myofibroblasts, no endothelial sproutings were observed. These observations strongly suggest that the formation of a new capillary network in vitro is substantiated by the intervention of myofibroblasts.

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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, as the effect of Vitamin B₆ on forming atherosclerotic lesion was not obvious, we examined the effect of Vitamin B₆ and its antagonists on the cultured endothelial cells from fetal bovine aorta. The patients with homocystinuria, who lack the activity of cystathione 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 sulfurcontaining amino acid metabolism might play a role in the formation of atherosclerosis in monkeys fed on Vitamin B₆ deprived diet. Because the patients with cystathionine synthetase deficiency had homocysteine in their blood at higher concentrations, we also studied the effect of homocysteine and its precursor, S-adenosyl-1-homocysteine.
We evaluated the effect of the above substances on the cultured endothelial cells with cell detachment assay by modifying the method of Harker et al. When seeded endothelial cells became confluent in 24 multiwell plate (Corning 25820), test media were added. After 24-hour incubation, each well was washed with PBS and still-adherent cells were counted. Results were expressed as percent detachment compared to control: % Detachment =(Control—Test)/Control×100.
We used isoniazid and deoxypyridoxine as Vitamin B₆ antagonists. When isoniazid was added, the results were 27.2±1.7%, 49.4±5.7%, 52.8±3.5% at concentration 1mM, 2.5mM, 5mM, respectively. When deoxypyridoxine was added, the results were 12.3±8.3%, 25.4±7.0%, 41.9±2.7% at concentration 0.5mM, 1mM, 2.5mM, respectively. On the other hand, we used pyriodoxal phosphate, active form of Vitamin B₆, and pyridoxal HCl as Vitamin B₆. When pyridoxal phosphate was added, the results were 0.9±3.6%, 4.4±2.5%, 8.1±2.2% at concentration 0.01mM, 0.1mM, 1mM, respectively. When pyridoxal HCl was added, the results were 7.5±1.5%, 11.7±4.1%, 18.9±2.6% at concentration 0.01mM, 0.1mM, 1mM, respectively. Also sulfur containing amino acids, DL-homocysteine and S-adenosyl-1-homocysteine was tested. When DL-homocysteine was added, the results were 2.8±6.4%, 19.0±6.3%, 16.5±6.3% at concentration 0.5mM, 1mM, 2mM, respectively. When S-adenosyl-1-homo-cysteine was added, the results were 24.2±9.1%, 32.9±7.3%, 33.2±8.1% at concentration 0.5mM, 1mM, 2mM, respectively.
The role of Vitamin B₆ deficiency in the formation of atherosclerosis is not clear in this study, which uses endothelial cells alone in vitro. However, it was interesting that Vitamin B₆ itself, as well as its antagonists, had endothelial detaching potential, although tested drug concentrations had to be taken into consideration. It was supposed that the atherosclerosis may be caused by the impairment of multiple factors, for example, not only endothelial cells but also smooth muscle cells, platelets, coagulants and their interactions in vivo. Vitamin B₆ depletion may also take part in the formation of atherosclerosis by influencing many factors but many problems regarding these mechanisms still remain to be clarified.

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β-VLDL observed in Type III-hyperlipemia (E 2/2 phenotype) is supposed to be one of the atherogenic lipoproteins because of high concentration of cholesterol. β-VLDL metabolism in arterial wall is not well studied. Therefore, we investigated the pathway of β-VLDL metabolism in cultured human arterial smooth muscle cells (SMC).
Human arterial SMC was cultured from the artery of operated stomach by the methods previously reported. VLDL and LDL from normal subject and β-VLDL from Type III-hyperlipemia (E 2/2 phenotype) were prepared ultracentrifugally. At first, we studied each I¹²⁵-labeled lipoprotein metabolism (cell binding, internalization and degradation) in SMC cultured with various concentrations of the unlabeled one. Secondly, the inhibition by unlabeled LDL on cell binding of labeled VLDL and β-VLDL was studied. Finally binding activity (B_max) of each lipoprotein was calculated on Scatchard plot.
Each unlabeled lipoprotein inhibited cell binding, internalization and degradation of each labeled one in dose dependent manner. Unlabeled LDL inhibited cell binding of labeled VLDL and, β-VLDL in dose dependent manner. B_max of β-VLDL was lower than others.
We concluded that (1) VLDL, LDL and β-VLDL were metabolized through the same receptor, probably “B·E receptor”, of SMC, (2) the lower affinity of β-VLDL to SMC might induce delayed catabolism and easy deposition of this lipoprotein in the aretrial wall.

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Effect of insulin on low density lipoprotein (LDL) metabolism in cultured human fibroblasts were studied in a serum-free system to avoid serum-effects which may interfere the hormone actions. Serum starvation increased LDL binding to the fibroblasts by 37% compared to non-treated cells, while pretreatment with lipoprotein deficient serum (LPDS) increased the binding more than three folds. LDL binding to the serum-depleted fibroblasts was saturable and cell concentration-dependent, furthermore LDL incorporation into the cells as well as LDL degradation by the cells were dependent on the incubation periods and the cell concentrations.
Insulin did not change the LDL pathway activity up to 2 hours, however 18 hour incubation with insulin increased the LDL binding, internalization, and degradation by 30-60% in the serum-starved cells, and approximately 10% in the LPDS treated cells. A half maximal insulin stimulation of the LDL pathway was observed at 5-10ng/ml insulin.
The results indicate that the serum-starved cultured fibroblasts can afford a system for the study of the mechanisms of hormonal regulation of LDL pathways, since this system is not interfered with various factors present in sera such as growth factors, and also suggest that insulin palys
a physiological role in the regulation of LDL pathways.

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We previously described that under hypoxic conditions esterified cholesterol and triglyceride accumulated in cultured cells. To elucidate the mechanism of lipid accumulation in hypoxia, we studied the effects of hypoxia on the synthesis of cholesterol and fatty acids in cultured cells. The incorporation of [¹⁴C]acetate into lipids of cultured rabbit skin fibroblasts was determined during hypoxic incubation (5%O₂) and control incubation (20%O₂). When fibroblasts were cultured in 20 normolipemic rabbit serum (NRS), the incorporation of [¹⁴C]acetate into cholesterol was decreased in hypoxia compared with control (15.0×10³ and 48.0×10³cpm/mg prot, respectively). When in 20% hyperlipemic rabbit serum (HRS), the incorporation was also decreased in hypoxia compared with control (1.2×10³ and 1.9×10³cpm/mg prot. respectively). Incorporation of radioactivity into total fatty acids was not increased under hypoxic conditions, nevertheless the esterification of radio-active fatty acids for triglyceride was increased remarkably in hypoxia compared with control in both 20% NRS (17.7×10³ and 6.4×10³cpm/mg prot.) and 20% HRS (19.8×10³ and 12.9×10³cpm/mg prot.) respectively. The effects of hypoxia in aortic smooth muscle cells were similar to that in fibroblasts. We concluded that the synthesis of triglyceride increased but that of cholesterol was inhibited under hypoxic conditions. The mechanism of esterified cholesterol deposition under hypoxic condition is remained.

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Serum apolipoprotein (apo) C-II and E levels were determined by a single radial immunodiffusion method in 84 type II diabetic patients, 27 males and 57 females. Serum apo C-II and E levels were high in diabetic patients, as compared with control subjects. Serum apo C-II and E levels in diabetic patients with type IIa, IIb and IV hyperlipidemia were higher than those in the patients without hyperlipidemia. Serum apo E level in diabetic patients with type IV hyperlipidemia was higher than that in the patients with type ha hyperlipidemia. There was no significant difference on serum apo C-II and E levels between diabetic patients without hyperlipidemia and control subjects. In diabetic patients, serum apo C-II, as well as serum apo E, positively correlated with plasma cholesterol (Ch), plasma triglyceride (TG), VLDL-Ch, VLDL-TG, VLDL-Ch/TG ratio, LDL-TG, HDL₂-TG or HDL₃-TG, and negatively correlated with LDL-Ch/TG ratio. In diabetic patients without hyperlipidemia, serum apo C-II and E levels in poorly controlled patients were higher than those in well controlled ones. In the same patients, serum apo C-II and E levels in the patients treated with insulin were higher than those in ones treated with diet only or oral hypoglycemic agents.
These results indicated that serum apo C-II and E levels in type II diabetes were altered by the metabolic change of TG-rich lipoprotein, and also by the grade of glycemic control or the kind of treatments.

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