In the present study, the interaction between the ultrastructure of arterial wall and blood components was investigated by scanning electron microscope in order to clarify the pathogenesis of vascular injuries.
1) Structural peculiarity of endothelium
Many active microvilli with a length of 7-20μ and a width of 0.1μ were seen on the surface of endothelium in cardiac ventriculus and original portions of aorta. Such long microvilli were not found on the endothelium of arteries distal from aortic arch, but a small number of microvilli with a length of 2-3μ were seen on these endothelial surface. These many microvilli on the endothelium of cardiac ventriculus and original portion of aorta may protect the vessel against damages caused by collision of blood components, because turbulent blood flow occurs at the portion.
2) Structure of marginal fold and its function
The different structure in various portions of aorta were also seen on marginal folds between the endothelial cells. In aortic arch and thoracic aorta, marginal folds appeared to be flexible. In these portions, leukocytes penetrated easily under the endothelial space through the marginal folds, while, in abdominal aorta, the structure of marginal folds were so tight that leukocytes were rarely found in subendothelial space. As described afterward, we have found that leukocyte penetrated under endothelial space and removed the deposited lipids in arterial wall. From these observations, it is considered that the atherosclerotic lesions develop in abdominal aorta more preferentially than aortic arch. Thus, the structure of marginal folds seem to relate to formation of atherosclerotic lesions.
Scanning electron microscopic study of cerebral arteries of rabbit indicated that swelling, dissolution and destruction of marginal folds between endothelial cells were induced by constriction of renal arteries, leading to formation of hole, which we named “wormy crater”. It is suggested that blood components leak through the crater to perivascular space, causing brain edema, angionecrosis or hemorrhage. These craters were also found in human cerebral arteries of autopsy.
Pathoanatomical examination of human autopsy showed that there were the structural differences in various portions of coronary arteries. In the original portions of coronary artery, marginal folds were seen on the boundaries of endothelial cells. On the other hand, in peripheral portions of the coronary arteries, marginal folds were not found and individual cells were separated by shallow grooves. It is considered that boundaries of endothelial cells in peripneral portions would not be affected by dissolution and destruction.
Such damage of marginal folds between arterial endothelium were seen in septicemia. We found that the endothelial cells became extinct after bacteria adhere to marginal folds. Thus, alternation of old and new endothelial cells are initiated from boundaries of cells.
Further, we found new type of endothelium, mamed as “jigsaw puzzle epithelium”, in artery of papillary muscle. This structure may be related to the function of cells, which contract and expand very widely.
3) Cellular components around endothelium and deposited lipids
From the examination of experimental hyperlipidemic animals, it is postulated that circulating leukocytes penetrate under the endothelial space and take the deposited lipids into self bodies. Then, the cells, containing lipid rich particles, go back to blood stream again. The deposited lipids in the atherosclerotic lesion would be removed by this process, which we named as “exotissuesis with lipid containers”. We presume that lipid containers, which could remove deposited lipids at cellular level, play important roles in regression and prevention of atherosclerosis.
We have subsequently examined the excretory mechanism of lipid containers, which were released to blood stream. Various sizes of l

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Aortas and coronary and renal arteries were investigated in 41 cases with collagen diseases and related conditions, consisting of SLE, progressive systemic sclerosis, Wegener's granulomatosis, dermatomyositis, Behçet's disease, PN, rheumatic arthritis and Buerger's disease. Extent and degree of arteriosclerosis of both aortas and coronary arteries in the diseases were higher than those in controls with neither collagen diseases nor hypertension. Small mural thrombi were frequently encountered in the aortas and coronary arteries. The soil on which the thrombi developed was edematous change of fibrocellular intimal thickening with slight lymphocyte infiltration or occasionally no lesion.
This observation may bring a conception forward that hypercoagulability, proven by clinical investigation, is more important than changes in vessel walls in thrombogenesis of collagen diseases. A high frequency of thrombosis can result in common involvement in arteriosclerosis.
A microscopic figures of arteriosclerosis accompanied by healed arteritis showed unusually marked destruction of the tunica media and internal and external elastic lamellae.
The majority of renal arterial lesions in cases with collagen diseases in this study was arteriosclerosis, except in a case affected by polyarteritis nodosa.
Degree of renal arteriosclerosis exhibited positive correlation with extent of glomerular capillary obstruction or destruction due to glomerulitis and with interstitial fibrosis of the renal cortex.

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In order to clarify the role of leukocyte in regression of atherosclerosis, cholesterol levels were determined in leukocyte of rabbit fed with a semisynthetic fat rich diet. Free cholesterol levels of rabbit were slightly increased by feeding of fat rich diet. On the other hand, levels of cholesterol ester, both C₁₆ and C₁₈ fatty acid ester, markedly increased. The cells containing sudan stainable particles were also found in arterial blood of hyperlipemic animals.
Furthermore, free cholesterol and cholesterol ester were found in human sputum, nasal mucus and tear, in which leukocyte and its degradation products may be excreted.
These results support our morphological observation that leukocytes remove lipids deposited in atherosclerotic lesion, and suggest that leukocytes play important roles in prevention and regression of atherosclerosis.

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Lipid composition of light lysosomal membranes in rabbit and human arterial wall was analysed. Following properties were observed in these fractions.
1) Concerning phospholipid composition, percentage of sphingomyelin was remarkably high and that of phosphatidylethanolamine was rather low.
2) Molar ratio of free cholesterol to phospholipid was unusually high.
Such alternation of lysosomal membranes may possibly involve in cholesteryl ester accumulation in arterial wall by the reduction of acid cholesteryl esterase activity or the mulfunction in fusion capacity of lysosomal membranes.

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Prostacyclin (PGI₂), a platelet aggregation inhibitor produced and released from the endothelial cells of the vascular vessels, is though to be concerned with the development of thrombosis and atherosclerosis. In the present experiment using rat aorta, we studied the influence of Ca⁺⁺ antagonists of the release of PGI₂.
The PGI₂ activity was measured by the inhibitory effect of ADP-induced human platelet aggregation. Ca⁺⁺ antagonists used are Diltilzem, Verapamil and Cinnarizine. The influence of Ca⁺⁺ ionophore A23187 and dibutylyl c-AMP was also studied.
As a result, Ca⁺⁺ antagonists were all shown to have an activity to increase the PGI₂ release from the vascular walls. When the aorta was treated with A23187 having an activity to raise Ca⁺⁺ concentrations in cells, the PGI₂ release was accelerated. However, the c-AMP treatment had no significant influence on the PGI₂ release.
From these findings it was surmised that the PGI₂ release-increasing activity of Ca⁺⁺ antagonists would not be via changes in the concentrations of Ca⁺⁺ or c-AMP. Then, phospholipase A₂ was inhibited with hydrocortisone and at the same time the aorta was incubated in a buffer supplemented exogenously with arachidonic acid, to which were added Ca⁺⁺ antagonists to study the influence of Ca⁺⁺ antagonists on the arachidonic acid metabolic pathway following the stage of phospholipase A₂.
As a result, Ca⁺⁺ antagonists increased the amount of PGI₂ produced and released from exogenous arachidonic acid. However, A23187 and c-AMP had no influence on this route.
As can be seen from the results above, Ca⁺⁺ antagonists—Diltiazem, Verapamil and Cinnarizinezine—increase the PGI₂ release from the vascular wall but this effect is observed in the arachidonic acid metabolis pathway following the stage of phospholipase A₂. Therefore it was suggested that the mode of action of these Ca⁺⁺ antagonists would be independent on changes in the concentration of Ca⁺⁺ and c-AMP.

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Recently, prostacyclin (PGI₂) has been considired to be a circulating hormone on the basis of its effect on the hemodynamics. In this report we studied on the PGI₂ generation of aorta during the development of hypertension and atherosclerosis in DOCA/salt hypertensive rats.
PGI₂ activity of rat aorta was assayed by the inhibition percentage of ADP induced platelet aggregation. This inhibitory activity was not observed in the aortic ring treated with Indomethacin or 15-hydroperoxy arachidonic acid. Besides, by means of the thin layer chromatography of incubation medium of aorta and ¹⁴C-arachidonic acid, it was indicated that the prostaglandin generated in aorta was mainly PGI₂. Therefore, this bioassay system is reliable for measurement of PGI₂ activity.
Systolic blood pressure and PGI₂ generation of DOCA/salt hypertensive rats rose up untill 2 weeks after the administration of DOCA and saline. At third week hypertension was established, but PGI₂ generation declined in comparison with second week. The administration of DOCA alone did not cause hypertension and did not affect on the PGI₂ generation for 2 weeks observation.
From these results, acceleration of PGI₂ generation of aorta at second week might be due to defense mechanism against hypertension, and decreased PGI₂ generation in the established stage of hypertension with the damage of vessel wall might indicate the derangement of this defense mechanism.

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We investigated the characteristics of fatty acid metabolism in rat aorta and brain microvessels. Brain microvessels were prepared by the modification of the method by Brendel et al. It was confirmed that brain parenchymal tissues were not microscopically and immunologically contaminated.
[1-¹⁴C] palmilate was much more incorporated into brain microvessels than into aorta, and it was mainly incorporated into esterified lipid in aorta and into cot in brain microvessels. In high cholesterol diet-fed rat aorta, [1-¹⁴C] palmitate, oleaste, linoleate and octanate were more incorporated into cholesterol ester compaired with controls while in brain microvessels they were not. Both enzyme activities of acyl-coA synthetase and acyl-coA: cholesterol acyltransferase increased in high cholesterol diet-fed rat aorta, however, such changes could not be seen in brain microvessels. These data show one of the mechanisms of formation of atheromatous lesions which are easily formed in aorta and rerely formed in brain microvessels. On the other hand fatty acid oxidation activities were much higher in brain microvessels than in aorta or heart or liver. Fatty acid oxidation activities in brain microvessels of spontaneously hypertensive rats (SHR) decreased compared with normotensive rats whereas the decrease was not found in aorta or heart. Fatty acid oxidation activies in brain microvessels of SHR decreased at the age of 16-29 weeks at which hypertension persisted, but they did not change at the age of 9 or 14 weeks at which hypertension was not completed. These results suggest that persistent hypertension may cause decreased activities of fatty acid oxidation in brain microvessels. Both activities of acyl-CoA synthetase and carnitine acyltransferase did not decrease in brain microvessels of SHR, suggesting that intramitochondrial oxidation activities might be decreased.

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The properties of phospholipase A₂ (Plase A₂) in rat arterial wall were studied using 1-palmitoyl-2-[1-¹⁴C] oleoyl-phosphatidylcholine (POPC) as a substrate. The Plase A₂ activity increased linearilywith the protein concentration upto 250mg/tube. It was also proportionate to the incubation time upto 120min. Km for POPC in the arterial wall was 0.067mM, that was almost equal to that in rat liver. The aorta had two pH optima at 4.0 and 6.2. With cell fractionation, the highest activity was located in the cytosol fraction, although among the particulate fractions the lysosomal and microsomal fractions showed the highest activity at pH 4.0 and pH 6.2, respectively. The measurement of Plase A₂ in various blood vessels, such as the thoracic aorta, the abdominal aorta, the brain microvessels and the mesenteric arteries, the aortas showed higher activities than the other vessels at pH 6.2. In the aorta, the abdominal aorta had five times higher activity than the thoracic aorta. In pathological conditions, Hypertensive rats (SHR) showed lower activity at both pH's than the controls. Cholesterolfed (for 20 weeks) rats had significantly higher activity at pH 6.2 than the controls. These results suggest that Plase A₂ activity is closely related to a condition where lipid accumulation may occur in the arterial wall. The mechanism of action of and the significance of Plase A₂ in the arterial wall remain to be further studied.

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Regulation of cholesterol esterase (CEase) in rat arterial wall was investigated comparing that of lipase.
Cholesteryl [1-¹⁴C] oleate sonicated with phosphatidylcholine was used as a substrate. The ratio of activity of 105000xg supernatant fraction to pellet fraction was different between CEase and lipase. This suggested that CEase was different from lipase in respect to the mode of existence in membrane. Both enzymes werereleased by 0.1% Triton X-100 from 105000xg pellet fraction. Acid CEase activity was decreased by heparin but neutral CEase was not affected. Acid and neutral lipase activities were slightly decreased by heparin. This finding also suggested CEase was different from lipase. The enzyme released during preparation (sup-I) was different from Triton released enzyme (sup-II) in respect to the affinity to heparin-Sepharose. When sup-I or sup-II was applied to heparin-Sepharose affinity chromatography, bound CEase was eluted by 0.5M NaCl solution. Neutral CEase activity was detected in bound fraction of sup-I. Lipase activity of sup-I was not detected in any fraction. Acid and neutral CEases were detected in bound fraction of sup-II. Acid lipase activity was eluted by 1.5M NaCl solution. These findings also suggested CEase was different from lipase. The effect of some substances such as VLDL, LDL, HDL and apoc-II on Acid CEase in unbound fraction was almost the same in sup-I and sup-II. And the effect on neutral CEase in unbound fraction was almost the same in sup-I and sup-II. This means that the enzymes which have no affinity to heparin were regulated under the same mechanism despite the different mode of existence in the cells. The effect of VLDL, LDL, HDL or apoc-II on neutral CEase in bound fraction was different between sup-I and sup-II. Neutral CEase activity in sup-II increased by lipoproteins and decreased by apoc-II. The enzymes which can bind to heparin have different regulation mechanism due to the difference in mode of existence.
Further investigation is needed for clarifying the role of these mechanisms in pathological conditions.

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Epidemiological and experimental investigations show that hypertension superimposed by hyperlipidemia accelerates atherosclerosis. However, the mechanism is not clearly understood. We have already reported in the previous paper that prolonged hypertension specifically decreased arterial cholesterol esterase (CE-ase) in SHR and SHRSP resulting in an accumulation of cholesterol and cholesteryl ester in the aorta. To extend these studies, the present investigation was undertaken to examine responses of the arterial enzyme to spontaneous hypertension superimposed by hypercholesterolemia. Effects of hyperlipidemia was examined by giving for 16 weeks either a synthetic diet containg 1% cholesterol and 0.5% cholic acid or the diet depleted of cholesterol to 10 week old normotensive female WKR. Effects of hyperlipidemia superimposed by hypertension was examined with female SHRSP treated likewise. Effects of hypertension superimposed by hyperlipidemia was examined with male and female SHRSP which were given for 16 weeks the cholesterol diet either with or with hypotensive drugs. Rat arterial CEase did not change by hyperlipidemia per se, but was elevated by hypertension superimposed by hypercholesterolemia. Accompanying the change in the enzyme activity aortic cholesterol content increased significantly. On the other hand, there were no differences in aortic CEase activity between hypotensively treated and nontreated SHRSP both hypercholesterolemia. This seems to be due to counteraction between the decreasing effect of hypertension and the increasing effect of hypercholesterolemia on arterial CEase. LPL and NAGA activities were elevated by hypercholesterolemia and hypertension, respectively. These results indicated that acceleration of atherosclerosis due to hypertension superimposed by hypercholesterolemia results from relative deficiency of CEase to increased infusion of serum lipids.

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Recently from our laboratory, it has been reported that serum nonspecific carboxyl esterase (tributyrin hydrolase) might be converted to lipase (triolein hydrolase) in aorta.
In the present report, the characteristic of tributyrin hydrolase in rat arterial wall were elucidated in order to clarify the mechanism of conversion from esterase to lipase.
Tributyrin hydrolase activity was estimated in homogenate of rat arterial wall using glyceryl tri[1-¹⁴C]butyrate as a substrate.
Optimum pH was found broadly from 6.0 to 9.0. Ca²⁺, Mg²⁺ or Ni²⁺ inhibited the tributyrin hydrolase activity by 20-30%. Zn²⁺ or Na⁺ inhibited the activity by 30-40%. Cu²⁺ inhibited the activity by 70%. Mn²⁺ or Ba²⁺ had no effect on the activity. HDL increased tributyrin hydrolase activity to the extent of 160%. VLDL or LDL or apo C-II or apo C-III had no effect on the activity.

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Effects of γ-oryzanol on the serum HDL, LDL and total cholesterol (ch) and Triglyceride were studied in 17 cases whose age was 47 years old to 82 years old (mean age was 71.2 years old). Three hundred mg/day of γ-oryzanol was given for 4 months.
Serum HDL-ch was 50.0±2.3 (m±SE) mg/dl before γ-oryzanol and 53.7mg/dl 3 months later but HDL-ch which was below 50mg/dl before treatment increased significantly (p<0.01) from 43.3mg/dl to 50.4mg/dl 2 months later. HDL-ch below 45mg/dl increased significantly (p<0.01) from 41.3mg/dl (mean) to 50.0mg/dl (mean) 2 months later. LDL-ch decreased significantly (p<0.01) from 158.8mg/dl to 134.8mg/dl after γ-oryzanol. HDL-ch×10/LDL-ch showed significant changes (p<0.01) (from 3.26 to 4.14) 2 months later. Total-ch did not change and between 203mg/dl and 208mg/dl. Triglyceride did not show significant changes.
It is suggested that γ-oryzanol alters the metabolism of HDL-ch and LDL-ch and increases serum low HDL-ch.

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HDL-cholesterol, HDL-phospholipids and their ratio (HDL-cholesterol/HDL-phospholipids) were determined in 55 patients with atherosclerosis before and after administration of pantethine. (600mg/day)
Five from above 55 patients whose HDL-cholesterol had increased predominantly in the above trial were once again given pantethine in the same dose for additional 1-2 months after a month withdrowal of medication. HDL₂ and HDL₃ were separated according to the simple procedure with the combination of lipoprotein precipitation in NaPhT-MgCl2 and ultracentrifugation. Phospholipids of HDL from five cases were separated on the silica gel G thin layer chromatograph.
Results were as follows,
HDL-cholesterol, HDL-triglycerides and their ratio were increased 3, 6 and 12 months after administration of pantethine. Lecithin was the major phospholipid which increased in HDL fraction of patients treated with pantethine. Subfraction of HDL revealed a high increase of HDL₂ and its constituents, that is, cholesterol, phospholipids and alpha-1-lipoprotein.
These results led us to the conclusion that pantethine may be useful for the prevention and treatment of atherosclerosis.

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The effect of ML-236B on serum cholesterol level was assessed in 21 patients with essential hypercholesterolemia on dosis of 15-90mg/day for 6-24 weeks. Reduction rates was compared in three groups divided according to their cholesterol levels; group A: 11 patients, more than 400mg/dl, group B: 5 patients, between 300-400mg/dl and group C: 5 patients, less than 300mg/dl.
Cholesterol level was reduced 33% in group A at the 16th week, 24% in group B and 20% in group C at the 12th week. The mean value of serum HDL-cholesterol level increased 17% at the 16th week after starting of the treatment. This effect was particularly significant in patients with the level less than 40mg/dl. Although X-ray follow up studies did not show any changes in the degree of thickend Achilles tendons, an apparent softening of the tendon was seen in one patient and ankle pain on motion was relieved in another patient. No changes were seen in triglyceride level during trial.
Physical and laboratory examination did not demonstrate any untoward effects of the drug during the observation period except for slight increases in GOT, GPT and CPK.

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The change on serum total cholesterol and triglyseride in 78 patients of essential hypertension with treatment of diuretics and antihypertensives has been observed at same month or at least same season for 1-12 years (mean 5 years).
1. In the patients treated with thiazide alone (1-2 Tab.) changes of total cholesterol and triglyceride levels in mean were from 205.8 to 213.8 and 11.6 to 119.6mg/dl respectively. These changes were not significant, however, elevation of total cholesterol over 30mg/dl and triglyceride over 50mg/dl was seen in several cases.
2. There were no significant difference in the incidence of vascular diseases in group with thiazide alone, compared as group with α-Methyl-dopa and other antihypertensives. Uncontrolled hypertension and abnormal elevation of serum lipid were considered as the main factor of vascular diseases.
3. Serum lipid should be measured repeatedly during long-term treatment with thiazide and other antihypertensives.

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Recently atherosclerosis in young people has been much discussed. Accordingly, we carried out a survey of degrees of obesity, serum lipid concentrations, uric acid (UA) concentration, and atherogenic index (AI) in primary school children (374 boys and 358 girls).
1) HDL-cholesterol (HDL-C) and UA concentrations of the boys were higher than those of the girls, while AT and triglyceride (TG) concentration were higher in the girls. There was no significant difference in total cholesterol (TC) concentration between the sexes.
2) Serum HDL-C was age dependent.
The value for girls became higher for the first time after the 5th grade than the value for boys, which suggests that HDL-C is related to the activity of gonad.
3) The risk factors of atherosclerosis were greater for boys of more than 20% overweight than for boys of other weight groups, while girls of more than 20% overweight did not have risk factors significantly different from those of the other girls.
4) Although there is a relationship between the degree of obesity and serum TG values in adults, there was no such relationships observed in the girls in this study. There was no relationship between UA and TG in both sexes. Therefore, we suggest that there may be differences in lipid and UA metabolism between adults and children.
5) We feel it is necessary to take a measure to prevent the degree of obesity in children (particularly in boys) which exeed the 20% overweight level.

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The separation of VLDL from the subjects of hypertriglyceridemia was performed through the heparin affinity column. Normally, the apolipoprotein composition in VLDL were apolipoprotein B (apo B), apolipoprotein E (apo E) and apolipoproten C (apo C), 37%, 50% and 13%, respectively.
VLDL recovered from the first peak of the column fraction was poor in apo E and rich in apo C (apo E poor VLDL) and was heparin unbound. VLDL recovered from the second peak of the column fraction was rich in apo E and poor in apo C. (apo E rich VLDL) and was heparin bound.
Incubation studies of these two kinds of VLDL with human skin fibroblasts revealed that apo E rich VLDL had higher association to the cells, binding to the cell surface, incorporation into the cells and degradation by the cells, comparing from apo E poor VLDL.
These facts suggested that VLDL of hypertriglyceridemia had another metabolic pathway into the cell except converting to the LDL. This speculation was compatible with the results of VLDL-B turnover study reported by Readon et al.
We expressed for apo E rich VLDL as VLDL_E after HDL_C reported by Mahley et al.
Mechanism of the higher interaction of VLDL_E to the cell will be concerned to the amount of apo E that has high affinity to the cell surface like apo B and to the low amount of apo C that might act as a inhibitor of the binding to the cell surface covering the binding site of apo B or apo E in the lipoprotein particle.

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Elastase (E), ELE-ES, 90U/mg, Eisai Co., was administered to experimentally induced hyperlipemic rabbits kept on 1% cholesterol (C) containing feed. A dose of 10mg/kg/day of E was administered i. p. to the hyperlipemic rabbits. Once increased serum lipids were not lowered in 2-4 weeks, but serum elastin decomposing and Suc(Ala)₃ NA hydrolyzing activities were both increased. Elevated serum lipids then showed tendency to be lowered in 6-8 weeks after E administration, and serum C level was significantly lowered. Serum elastin decomposing and Suc(Ala)₃ NA hydrolyzing activities were not altered at this period. Correlation between time-course change of these serum enzyme activities and amount of tissue lipids, elastin and other components of the connective tissue was then examined. C content in tissue increased after 10 weeks of the C loading, and C in the liver was significantly lowered by E administration. Hydroxyproline content in tissues increased after C loading, and significant lowering effect was induced by E in the lung where C content was significantly higher after the C loading. It might be considered that E improves significantly the metabolisms of lipids and the connective tissues in the hyperlipemic conditions.

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