McCully et al reported that the arteriosclerotic plaques were found in the aorta and arteries of rabbits given homocysteine thiolactone, methionine or homocysteic acid parenterally, and McCully et al reported that pyridoxine prevented thrombosis and pulmonary embolism in rabbits given homocysteine thiolactone or methionine but did not prevent arteriosclerotic plaques. Also, there are reports that abnormalities of homocysteine metabolism are associated with arteriosclerosis and vascular thrombosis.
In this study authors observed that pyridoxal phosphate (active form of pyridoxine) can prevent the arteriosclerotic plaques of rabbits given I-methionine parenterally. Furthermore, authors demonstrated that 1-methionine or dl-homocysteine itself aggregate the human platelets in vitro and enhance the platelet aggregation by ADP or noradrenaline in vitro. The similar results were observed in rabbit (in vivo).
From these studies, authors reconfiremed Mc-Cully's reports that sulfur-contained amino acid induces fibrous plaques in rabbits and observed that pyridoxal phosphate inhibits the formation of the fibrous plaque of arteries in rabbits given sulfur-contained amino acid and the arteriosclerogenecity of sulfur-contained amino acid is resulting from the increasing platelet adhesiveness or agregation.

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The morphologic changes of endothelial cells in the descending aorta were studied with scanning electron microscope (SEM) on hypovitamin C guinea pigs with or without cholesterol added diet for 4 weeks. During experimental period, serum cholesterol level and permeability into the aorta with labeled cholesterol were estimated. Four groups of experimental animals were fed on ordinary diet (group 1), on ascorbic acid deficient (group 2), on cholesterol added ordinary diet (group 3), on ascorbic acid deficient and cholesterol added diet (group 4). Endothelial changes observed with SEM were shown in Fig. 1. Flattering of the endothelial cells with indistinct cell border were observed in the early phase of hypovitamin C. Swollen endothelial cells with disarrangement were pild up and formed atherosclerotic lesion in the chronic hypovitamin C. Atherosclerotic changes were appeared in the early period of the experiment in group 3 and 4. Most characteristic feature in the group 4 was separation of endothelial border. Serum cholesterol of the group 3 and 4 were significantly elevated as compared that of the group 1 and 2 except the first week of feeding. Hypercholesterolemia indused with cholesterol feeding was enhanced with hypovitamin C (Fig. 2). The contents of 4-¹⁴c-cholesterol were measured 3rd, 7th and 14th day after intraperitoneal administration to the animals of the the second experimental week. The count in serum of the group 2 and 4 was appearently increased as compared with the other groups at 3rd and 7th day. Uptake of the aortic wall at the 3rd day of the group 4 was significant higher than the other groups. There was no difference of the uptake between the four groups at 7th and 14th day (Fig. 3). These findings suggest that injury of the endothelial cells induced with hypovitamin C is important one of initiaiing factors of atherosclerosis, and hypercholesterolemia and enhanced permeability of arterial wall acts as promoting factors of the atherogenesis.

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We have analyzed fatty acid composition of the aortas, hearts and adipose tissues of the mice treated with cigarette smoke of long-term inhalation. We have observed two characteristic factors: (I) Linoleic acid percentage in aortic cholesterylester fraction and phospholipid fraction decrease by smoking (2) Arachidonic acid percentage in aortic cholesterylester fraction decrease. These findings mean that smoking brings about an atherosclerogenic condition in the aorta.

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It is well described process that accumulation of arterial lipids, especially cholesteryl ester (CE), occurs during aging and atherogenesis. In addition, it has been reported that the activity of CE hydrolase (CEH) in aorta is lower in species susceptible to atherosclerosis (rabbit, swine etc.) than in the resistant (rat etc.). Accordingly, the arterial enzymes relating to lipid hydrolysis and synthesis are postulated to play important roles in atherogenesis.
The present investigation was undertaken to clarify the mechanism underlying the accumulation of lipids in arteries during aging and atherogenesis using SHRSP, SHR and WKR which were maintained on lab. chaw for 1, 6 and 11 months after weaning. For this purpose, the following observations were made 1) the change in the level of arterial lipids, 2) the change in the arterial lipid hydrolase activities [CEH, triacylglycerol hydrolase (TGH) and lipoprotein lipase (LPL)] and 3) the change in the membrane stability of lysosomes containing CEH and TGH.
The difference in blood pressure between SHRSP and SHR or SHR and WKR was approx. 20mmHg at 1 month, and approx. 50mmHg at 6 and 11 month, in the increasing order of SHRSP, SHR and WKR. The aortic cholesterol level progressively increased during aging. The degree of the increase was the greatest in SHRSP. The level paralleled with blood pressure. Lysosomal CEH and TGH activities greatly decreased during aging. The activities at 1 month after weaning were SHRSP>SHR>WKR. On the other hand, non lysosomal LPL activity was unchanged during aging. Since lysosomal membrane is known to be sensitive to aging, the stability of liver lysosomal particles was measured by the leakage of CEH and acid phosphatase. The stability of lysosome significantly decreased due to aging in all strains, and the pattern of the decrease in the stability resembled that of the loss of aortic CEH activity.
These results suggest that the accumulation of arterial lipids during aging and atherogenesis results at least partly from the decrease in activity of lysosomal lipolytic enzymes as a consequence of labilization of the lysosomal membrane.

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1. A rapid and sensitive method to detect lysosomal lipolytic activity (MU-oleate hydrolase) was established in rat liver using 4-methylumbelliferyl oleate as substrate. In order to detect the activity, neither phospholipid nor detergent was required and optimum pH was observed at 5.0.
2. MU-oleate hydrolase appeared to be similar to acid cholesteryl esterase. The results indicated that the enzyme might be in lysosomes, especially binding on lysosomal membrane, according to several kinds of subcellular fractionation techniques.

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1. Lysosomal MU-oleate hydrolase (MUH) from rabbit liver was partially purified by Sephadex LH-20, DEAE Sephadex A-50, Bio-Gel A-5m, hydroxyapatite column chromatography.
2. The enzyme was compared with other lipolytic enzymes such as acid cholesteryl esterase (ACE), triglyceride lipase (TGL) in this paper. The heat-sensitivity and other properties of both MUH and ACE were very similar.
3. MUH was activated by lysosome-rich fraction and the structure of lysosomal membrane might require for the capacity of activation.
4. MUH activity against enzyme concentration exhibited sigmoidal curve which suggested that MUH might be membrane associated enzyme and it might be activated by itself.

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Effect of chondroitin polysulfate (CPS) on the vascular cells in the arterial wall and heart muscle cells was studied in the state of hypertension. Twenty four Wister male rats were divided into four groups: 1) Control, 2) CPS, 3) hypertension, 4) hypertension plus CPS.
Hypertension had been initially induced by the method of Page. CPS was perorally administered at the rate of 6mg/100g body weight. Rats were then sacrificed at 2 and 4 weeks after the CPS treatment.
Increased ratio of ³H-thymidine-incorporated cells in the aorta and heart tissue was observed in the hypertension group. CPS-treated group tended to decrease of ³H-thymidine incorporated cells in the aorta and heart tissue in the hypertension state.
The histological study of the aorta showed the irregular arrangement of the elastica: nuclei were swelled, rounded and they approached each other. In the heart, small capillaries showed thickning of the media and hyaline degeneration in the heart muscle cells. Significant improvement was not observed by the CPS-treatment in histologically. Fibroblastic cells increased in hypertension group. But increased number of cells was suppressed by the CPS treatment.
A few of ³H-thymidine incorporated cells were marked in the blood of the CPS-treated rats, but not in the control group.
These data suggested that (i) cell mitosis was susppressed by the CPS treatment and (ii) CPS acted as the defence mechanism of body mesenchymal on the heart and arterial cells.

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The purposes of the present study are to elucidate the mechanism of the anticoagulation caused by aortic chondroitin sulfates (CS) and to compare anticoagulant activities of CS derived from the normal and sclerotic aorta.
CS extracted from the intima and media of the normal and sclerotic aorta obtained from Japanese autopsied cases were purified. Chemical analysis of the CS revealed that there was almost no difference in the per cent composition of CS-4-S, CS-6-S and dermatan sulfate and the sulfur content but the calcium content between normal and sclerotic CS. CS obtained were able to be completely digested to disaccharides by the chondroitinase ABC. The concentration of the CS examined in the coagulation studies was mimiced to the one in the aortic tissue.
The formation of the fibrin clot after admixing thrombin with plasma was inhibited by the addition of both normal and sclerotic CS (Anticoagulation). The latter showed less potent inhibitory effect on the formation of the fibrin clot than the former.
CS did not affect either hydrolysis of tosyl arginine methyl ester (TAME) or proteolysis of fibrinogen by thrombin in the absence of antithrombin III (ATIII). In the presence of ATIII, both normal and sclerotic CS accelerated the action of ATIII to inhibit the activity of thrombin to the same extent (Heparin-like action).
Both normal and sclerotic CS accelerated the aggregation of monomeric fibrin to make clot after neutralization from acidic solution (pH 5.3) to pH 7.3 (Acceleration of aggregation). Sclerotic CS accelerated the aggregation much more than normal CS did. Although the possibility appeared that calcium ions bound abundantly to sclerotic CS might exhibit stronger acceleration of the aggregation than normal CS, it could be ruled out by the fact that the further decalcification of the sclerotic CS by the ion exchanger resin, Amberlite IRC-50, could not affect the accelerating effect on aggregation at all.
It is possible to conclude as follows, (1) CS of aortic intima and media possess the anticoagulant activity derived from the accelerating effect on ATIII, (2) sclerotic CS exhibit weaker anticoagulant activity probably because of the accelerated aggregation of monomeric fibrin to make clot, and (3) weaker anticoagulant activity of sclerotic CS might contribute to the deposition of fibrinogen derivatives in the sclerotic lesion of the aorta.

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The aim of the present study was to investigate the effect of age on the property of collagen, the solubility and platelet aggregation activity, prepared from human aorta.
The tunica media prepared from 8 females aged 13 to 91 years was repeatedly extracted with 0.5M NaCl containing 0.02M EDTA pH 7.4, 0.5M acetic acid, 5M guanidine in 0.1M Tris-HCl pH 7.4 and hot 0.1M NaOH (98°C, 45min.) in this order.
To estimate collagen content of each fraction both hydroxyproline and hydroxylysine were determined, since hydroxylysine was an amino acid specific for collagen. Percent distribution of both amino acids among fractions was in good agreement with each other.
The proportion of neutral salt-, acetic acid-and guanidine soluble collagen to the total collgaen decreased with advancing age, whereas insoluble collagen increased.
Elastin was prepared from hot 0.1M NaOH residue by dithiothreitol reduction and was subjected to amino acid analysis. No significant differences were found among young and old individuals.
The insoluble collagen fraction was prepared from 5M guanidine residue by treating with 0.5% deoxycholic acid followed by homogenization in cold saline. The relative platelet aggregation activity of this fraction gradually decreased with age, suggesting that the insoluble collagen had undergone a further structural change.
These observations indicate that (1) the stepwise extraction enables to correctly estimate the agerelated decline in solubility of collagen and (2) the insoluble collagen would become less potent with age in a biological activity to induce platelet aggregation.

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Although there have been some reports claiming that the reduced glucose tolerance would be involved as a risk factor to IHD, we have earlier reported that the reduce of glucose tolerance is not always an important factor to IHD, but rather the abnormal pattern of FFA decrease during glucose loading would play a role as the accerelating factor to the occurrence of IHD which can not be neglected. In the present report, the effects of PGE₁ upon the carbohydratelipid metabolism to 50g-OGGT were investingated in relation to the pathophysiological mechanism of the Type B-FFA pattern as seen in the patient of IHD. In 20 cases of the patients with IHD showing the Type B-FFA pattern, PGE₁ (0.6μg/min.) with physiological saline 250ml was continuously infused into the right cubital vein for 180min. simultaneously with the start of 50g-OGTT, FFA, IRI and sugar in the venous blood sample were measured before, 30, 60, 90, 120 and 180min. after glucose loading. These biochemical variations were comparatively evaluated with the variations observed when PGE₁ was not administered. As a result, the following were found out:
1) The effects of PGE₁ upon the Type B-pattern of FFA may be elucidated by that the Type B was changed into Type A following administration of PGE₁. Specifically, the FFA values without administration of PGE₁ were significantly lowered respectively to 0.009±0.05 and 0.09±±0.04 at 120 and 180 minutes after the loading in comparison of 0.45± 0.31 prior to the loading. On the other hand, the FFA value with PGE₁ administration amounted to 0.29±0.17 at 180 minutes after loading against 0.43±0.22 which was before loading, showing no signicant difference and the recovery of the oncedropped FFA level in blood into the prior level in 180 minutes after loading. The FFA values at 90, 120, and 180 minutes with administration of PGE₁ were significantly higher that those without administration of PGE₁.
2) In terms of there lationship between FFA_f and FFA_90′ or FFA_180′ no significant correlation was found between the two when PGE₁ was not adimistered, and the drop in the blood FFA during GTT varied with independence on the values prior to the loading, whereas, with administration of PGE₁ significant positive correlation was not between the two, with a correlation coefficient of r=0.865 and 0.885 with P<0.001. After loading, the blood levels of FFA varied with dependence on the values prior to the loading.
3) In the terms of the effects of PGE₁ upon the IRI level in the circulating blood the insulin secretion during the GTT was inhibited when PGE₁ was administered, against the values without administration of PGE₁. Specifically, the IRI_30′ and IRI_60′ with administration of PGE₁ were respectively 24±14μU/dl and 35±20μU/dl, whereas the counterparts without administration of PGE₁ were 47±30μU/dl and 56±36μU/dl indicating significant decrease and also a trend that the IRI_90′ IRI_120′ and IRI_180′ were lowered by the administration of PGE₁.
4) No effect was observed upon the glucose loading by the administration of PGE₁ and there was no significant difference between the blood sugar levels during the loading with administration of PGE₁ and the blood sugar levels without administration of PGE₁.
Conclusion: On the basis on the above results, a mechanism was assumed to exist that the decsease of FFA in blood in the patients with IHD would be abnormally delayed due to the excessive secretion of insulin against GTT, and it was also suggested that the overscretion of insulin

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The hypocholesteremic effect of dietary administered Chlorella was studied in rats.
Wistar male rats were fed on a hypercholesteremic diet containing 1% cholesterol and 0.25% cholic acid for seven days. Dried Chlorella was added to the hypercholesteremic diet 2-10% levels. Total serum cholesterol level of the cholesterol diet group was about 4 fold of the basal diet group, and this elevation depended on esterified cholestetol rather than free. This elevation of both types of serum cholesterol level was depressed by administration of Chlorella, while it was not effected to liver cholesterol level of hypercholesteremic rats.

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Lipoprotein pattern observed by disc electrophoresis in normal rats and dogs is characterized primarily by α-lipoprotein, showing difference from that in humans and monkeys.
Feeding rats with 1% cholesterol, 0.2% sodium cholate and 5% olive oil mixed in diet and 0.02% solution of 6-propyl-2-thiouracil in drinking water caused hyper-β-lipoproteinemia which developed a similar pattern to that of human hyperlipemia. Clofibrate was found not to be effective in reducing serum β-lipoprotein in the said experimental model, while lowering lipid levels in alimentary hyperlipemic rats.
A newly synthesized compound, Y-9738 [ethyl 2-(4-chlorophenyl)-5-ethoxy-4-oxazoleacetate] exhibited a potent lipid lowering effect in alimentary hyperlipemic rats. The compound was seven times as potent as clofibrate in lowering serum cholesterol levels. Also in the above animal model, Y-9738 not only lowered total lipid levels but also reduced serum β-lipoprotein and improved β/α ratio.

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Extensive atherosclerotic lesions of coronary artery and aorta, that is, fatty streaks, intimal thickening, cellular and fibrotic proriferation, “foam cell” accumulation, fragmentation of elastic lamina, calcification and presence of intercellular and extracellular fat deposition were induced within 6 weeks in rats fed on basal ration suplemented with 2% cholesterol, 0.5% cholic acid, 10% lard, 5% cain sugar and 0.2% methylthiouracil as atherogenic agent in addition to oral administration of 35 million U. S. P. units of vitamin D₂ per Kg body weight for the initial 4 successive days.
Vascular changes on early stage of this model, such as degeneration and fragmentation of elastic fiber, edematous reaction in nitima and calcification of media were mainly attributable to vitamin D₂-treatment because of lacking in significant rising of serum level of lipid at this stage. Atheromatous lesions developed after 2-4 weeks, accompanying with hypercholesterolemia induced by atherogenic diet.
The protective effect of pantethine, clofibrate, furazabol and their combination were studied on serum lipid levels and the 6th week atherosclerotic lesions in the coronary artery and aorta.
Pantethine (50 or 200mg/Kg/day) effectively prevented progression of the atheromatous lesion without considerable lipid lowering action by itself and pronouncedly displayed anti-atherosclerotic action in corporation with furazabol.
Clofibrate (100mg/kg/day) failed to protect the atheromatous lesions in this model.

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The development of atherosclerosis is characterized by the focal accumulation of lipid in arterial wall. In this accumulated lipids, the content of cholesterol ester and phospholipid are high and triglyceride is not high. In order to clarify the mechanism contributing to this characteristic lipid content, we have undertaken an investigation of lipid metabolism concerned with cholesterol ester, phospholipid and triglyceride in rat arterial wall. Furthermore, the effect of pantethine, as an antiatherosclerotic agent, on the lipid metabolism was investigated. Male Wistar-King strain rats were fed a normal or a high cholesterol diet with or without pantethine for 4 weeks in the following manner: 1) Normal diet, 2) Normal diet plus pantethine (200mg/day), 3) High cholesterol diet which contains 1% cholesterol-0.5% cholic acid and 4) High cholesterol diet plus pantethine. Increased of acyl-CoA synthesis from palmitate-¹⁴C was observed when extracts of rat arterial wall were incubated with pantethine (1mg/ml). Cholesteryl ester synthesis from palmitate-1-¹⁴C in the arterial wall slices was higher with arteries from rat on high cholesterol diet than with those from rats on normal diet, but the synthesis was reduced in the arteries of rats on high cholesterol diet with pantethine. Triglyceride synthesis from palmitate-1-¹⁴C was higher with arterial wall slices of rats on high cholesterol diet than with preparations from rats on normal diet and was increased with those of rats on high cholesterol diet plus pantethine. Phospholipid synthesis from palmitate-1-¹⁴C was higher with arterial wall slices of rats on high cholesterol diet than with those from rats on normal diet and was not reduced with those of rats on high cholesterol diet plus pantethine. From these results, pantethine might possess the preventive action of atherosclerosis by reduction of cholesterol ester synthesis and no reduction of increased phospholipid synthesis.

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