Four humdred and twnety arteriosclerotic patients with or without hyperlipidemia have been followed up for seven years epidemiologically. It has been clearified that cerebral infarction and ischemic heart disease have been found with higher incidence in the hyperlipidemic group.
Intimate correlation in the incidence have been found between hypercholesterolemia and ischemic heart disease and between hypertriglyceridemia and cerbral infarction.

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Effective β-adrenergic blocking agent propranolol was administered for eight weeks (60mg daily for initial seven weeks and 120mg for successive one week) to investigate its action on plasma lipids and lipoprotein metabolism in patients with atherosclerosis. Subjects selected for studies were each five male and female post-apoplectic inpatients with normal plasma lipids.
Plasma FFA was gradually but significantly elevated with propranolol following initial slight reduction. Incorporation of U-¹⁴C-glucose into plasma total fatty acid and cholesterol fractions were also accelelated significantly. Reduced β-oxidation and reesterification of fatty acid and increased fatty acid synthesis may be responsible for these effects of propranolol. Elevation of triglyceride and total lipids of VLDL and concomitant reduction of LDL and HDL were observed. Apolipoprotein VLDL, however, remained stable and apolipoprotein LDL and HDL decreased. FFA release with heparin and postheparin lipolytic activity were apparently suppressed with propranolol.
These results suggest that propranolol inhibits the conversion of VLDL to LDL and probably to HDL through inhibition of lipoprotein lipase activity. Examination about the effect of propranolol on the synthesis of lipoprotein lipase, its release into blood stream and on its enzymatic activity is in progress.

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Nicomol [2, 2, 6, 6-tetrakis (nicotinyloxymethyl)-cyclohexanol], a derivative of nicotinic acid, was given to 25 patients with hyperlipidemia of type II_a, II_b or IV at a level of 0.6-1.2g per day for 8-32 months. Facial flush, erythema and itching of the skin temporarily occurred as side effects in 11 patients. Lowering of the plasma cholesterol level more than 50mg/100ml was attained in 35-50% of the cases. Decreases in plasma triglyceride concentration were observed in almost all of the cases of type II_b and IV in which the triglyceride level before treatment was more than 150mg/100ml. However, in several cases in which the triglyceride level was previously normal, that is, in type II_a hyperlipidemia, plasma triglyceride showed a slight or moderate increase and there was a change in lipoprotein lectrophoretic pattern from type II_a to type II_b or IV.
In three cases of alcoholics (one female and two males) which presented type II_a or II_b hyperlipidemia before treatment, a sever hyperlipemia of type IV or V appeared during the course of the treatment. Prohibition of alcohol brought a prompt healing and the withdrawal of the drug also resulted in a recovery of plasma lipid level.

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Out of three free-living populations, 320 subjects over 40 years of age were randomly selected and divided into two groups: the group treated daily with 0.75mg of an anabolic steriod, ethylnandrol, and the control group on placebo (Table 1). Their physical status, ocular fundus, ECG, urine and, on fasting blood sample, serum cholesterol, triglyceride and GOT were examined once or twice a year for five years. At every follow-up observation, the possible episode of ischemic disease and the side effects of the treatment were examined. At the 48th month examination, plasma fibrinogen and euglobulin lysis time were measured for the both groups.
The mean cholesterol levels of the treatment group were significantly lower than those of the control group throughout the observation period, although both groups had a seasonal fluctuation in cholesterol level high in summer and low in winter. The mean triglycerides were also lower in the treatment group (Fig. 1). Hypertension, defined as blood pressure above 160mmHg systolic and 95mmHg diastolic, decreased in the prevalence in both groups, and no difference is seen between the two groups. The mean concentrations of plasma fibrinogen were 266±58mg% for the treatment group, and 243±58mg% for the control group, the difference between the two being not statistically significant. The mean euglobulin lysis time was 342±45min. for the treatment group, 373±50min. for the control group. The euglobilin lysis time was significantly shortened in the treatment group. (p<0.05).
The primary episode of 6 cerebral infarctions occurred in the treatment group, while those of 6 cerebral infarctions and 2 myocardial infarctions in the control group. No death occurred in the treatment group, while 2 out of 6 cerebral infarctions died within one month of the onset of the disease in the control group (Fig. 2).
The three major risk factors for the cerebral infarction were age, hypercholesterolemia, and systolic hypertension. The risk factors for myocardial infarction were not yet evaluted since the numbers of patients were still too small.
No such side effects of long term administration of ethylnandrol as maleness, body weight gain, glucose tolerance and liver dysfuncton were seen in the subjects taking ethylnandrol for 5 years.

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Mural thrombosis and serum lipid levels have been known to play a part in the pathogenesis of atherosclerosis. The present study was under-taken to elucidate the relationship between serum lipid levels and blood coagulation in the nephrotic syndrome. In coagulation profiles, the partical thromboplastin times and the prothrom bin times were variable. The thrombin times were prolonged in general and proportional to the levels of total serum cholesterol; on the other hand, plasma fibrinogen contents, prothrombin and plasma factor V activities were generally increased. There were positive correlations between plasma factor V activities and the levels of total serum cholesterol, triglyceride and β-lipoprotein. Euglobulin clot lysis times as well as serial thrombin times showed a tendency to prolong, and thus the results were contradict each other in fibrinolysis. There were no relationships between serial thrombin times and the levels of serum lipids, but the administration of glucocorticoids. Fibrin degradation products in blood were almost within normal limits. There were marked increases of composed fatty acids of total serum lipids in nephrotic syndrome.

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In an attempt to clarify the alteration of lipid metabolism in the aorta of diabetes, aortas from rats with streptozotocin-induced diabetes were incubated in vitro with ¹⁴C-glucose and the incorporation of glucose into the lipid fractions was studied. Furthermore, triglyceride, diglyceride and monoglyceride content as well as the activities of lipase and monoglyceride hydrolase in the aorta of diabetic rats were measured. In diabetic rats, in which streptozotocin (80mg/kg) was given 4 days before sacrifice, the incorporation of glucose into the aortic triglyceride was significantly decreased when compared with the controls. Triglyceride and diglyceride content in the aorta of diabetic rats was significantly smaller than those of the controls. However, no difference in the monoglyceride content was found between these two groups. Although the aortic monoglyceride hydrolase activity was significantly decreased in the diabetic rats, there was no decrease in the lipase activity in these rats. These results suggest that the synthesis of triglyceride in the aorta is decreased in diabetic rats.

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In the genesis of atherosclerosis, the deposition of lipids in the arterial wall seems crucial. Acid Mucopolysaccharides (AMPS, glycosaminoglycans) are assumed to be involved in this lipids precipitation process. Recent studies suggest that the analyses of interstitial substances of many different tissues besides artery may be valuable to elucidate the atherogenetic process.
In man, AMPS content of the renal papilla is rich and comparable to that for the aorta, which seems to justify a detailed study on AMPS and lipids content of the renal papilla with age.
20 human kidneys of various ages from 1 to 78 were obtained at post mortem examination 4-12hr after death. None of the kidney used were involved by any evident disease process except nephrosclerosis. The main renal artery and papillary region were removed and analysed for AMPS and lipids (total cholesterol and phospholipids).
In the artery, AMPS and lipids levels increased with age. But in the papilla, AMPS increased during first five decades and decreased thereafter, while cholesterol content decreased until 50 years, followed by an increase.
Analysis of AMPS composition suggests that not only dermatan sulfate facilitates, but also hyaluronic acid might prevent the lipid precipitation in the renal papilla.

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Seven patients of acute myocardial infarction in convalescence were examined before, during and after Master's double two step test using radioelectrocardiography in order to predict prognosis. A typical case suffered from anterior infarction whose clinical course had shown an excellent recovery was illustrated. In this case in an early stage of recovery the ST segment elevated and the T wave inverted before exercise; during and after exercise the elevated ST segment shifted a more upper level and the inverted T wave deepened. Later on the next stage ST segment elevated and T wave shallowed or converted to positive tentatively after exercise. In the final stage of recovery ST segment was near the baseline and showed little elevation due to exercise as well as T wave unchanged. Out of the other six cases, 5 cases were examined sequentially. Three cases with fine recovery showed radioelectrocardiographic findings due to exercise resembled to those of the illustrated case. In another case the sequential exercise radioelectrocardiograms had shown no recovery. This case had an episode of relapse in the chronic stage. In summary the sequential changes of exercise electrocardiograms are resembled to the sequential changes of the resting electrocardiographic findings of acute myocardial infarction.

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Since vitamin C, ascorbic acid is essential for maintenance of the architecture and connective tissue metabolism of the aorta, the effects of chronic covert vitamin deficiency on serum lipids and the aorta were studied.
Male guinea pigs weighing about 300 grames were fed with scorbutic diet and supplemented 5mg of vitamin C twice a week to protect overt ascorbic acid deficiency. The control were fed with the same diet and administered 50mg of the vitamin on every other day. At the end of 8 weeks feeding, the animals were sacrificed after 24 hours fasting. Serum triglyceride was elevated in the deficient as compared with the control. In 66% of the deficient, white patchy plaques were observed in the arch and proximal portion of the aorta. Microscopic examinations revealed that the lesions were composed of fibrous thickening of the intima with lipid accumulation and degenerative changes in the middle of the media accompanied by deposits of lipids, mucopolysaccharide and calcium as shown in Fig. 1. Chemical determination and histochemical demonstration of hydrolases activities in the aorta revealed elevated activity of esterase, lipase, acid phosphatase and β-glucuronidase in the proximal portion of the aorta (shown in table 2).
These findings suggest that chronic ascorbic acid deficiency is a contributing factor in atherogenicity as shown in Fig. 2.

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Robbins reported the existence of Factor XIII and thereafter many investigators have reported the role of factor XIII in coagulation mechanism. This enzyme has, also, some actions on atherosclerotic changes in arterial intima. In order to clarify the relation between the enzyme activity and the atherosclerotic severity, we measured the activity of factor XIII and transglutaminase in human aortas.
Materials and methods
A) materials: Relatively fresh 10 human aortas were selected from forensic autopsy. The intima-media preparation was made from aortic arch, thoracic and abdominal aorta.
B) measurement of factor XIII: Partigen method.
C) measurement of transglutaminase activity: Modification of Dvilansky et al.
Results
We found both factor XIII and transglutaminase activity in each stages of atherosclerosis. Factor XIII seemed to be higher, but there were no significant increases. In transglutaminase activity, however, there was lower activity in stage 4.
Conclusion
In early stages of atherosclerosis, we found the some degree of enzyme activity. Laki et al reported the same result in animals, but they could not ascertain whether they were dealing with the tissue or the plasma enzyme. We also imagine that the large parts of enzyme in arterial wall may come from blood plasma, but some parts of it come from tissue itself. Factor XIII may have the important role in the dynamic balance of fibrinogen-fibrin system both in blood plasma and in arterial walls.

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Fibrin stabilizing factor (FSF) catalyzed intermolecular crosslinking of fibrinogen. Differing from the known zero-order relationship in fibrin crosslinking, the rate of crosslinking of fibrinogen depended on its concentration (first-order). When the concentraction was extremely high, FSF rapidly catalyzed crosslinking to produce gelation of fibrinogen. This observation suggests that filtrational condensation of fibrinogen may contribute to deposition of insoluble fibrinogen in atheromatous tissue.
In studies on mixtures of fibrinogen and fibrin, FSF showed almost equal affinities to both substrates; and,
a) at low concentration of fibrinogen, in which crosslinking of fibrinogen proceeded very slowly compared to fibrin, fibrinogen inhibited the stabilization of fibrin clots by competitively binding to FSF.
b) at high concentration of fibrinogen, in which fibrinogen could solubilize small amount of fibrin by forming the soluble fibrinogen-fibrin complex and inhibit fibrin clot formation, stabilization of soluble fibrinogen-fibrin complex by FSF added further inhibition of stabilization of fibrin clots. These findings may suggest the mechanism in which fibrinogen inhibit both polymerization and crosslinking of fibrin being produced in the circulating blood.

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Deposition of PAS-positive materials and thickenning of basement membrane are characteristic findings in diabetes mellitus, suggesting altered metabolism of glycoprotein.
Activities of β-glycosidases, includingβ-N-acetylglucosaminidase, β-N-acetylgalatosaminidse, β-galactosidase, β-glucuronidase and β-glucosidase were measured in intestine, liver, kidney and spleen of streptozotocin diabetic rats. Those enzyme activities except β-glucosidase were lowered specifically in diabetic kidney with a reciplocal increase in activity in serum. These were corrected by insulin treatment. Column chromatographic studies of β-N-acetylglucosaminidase revealed one of the fractions, the main fraction of kidney enzyme, was insulin dependent to maintain its activity. It is most likely that the decrease in activities of glycosidases in diabetic kidney might induce retarded degradation of glycoprotein which could be one of the causative factors of deposition of glycoprotein in diabetic microangiopathey.

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Elastin was isolated from the thoracic aorta of humans of different ages, sclerotic and nonsclerotic, and studied qualitatively and quantitatively. Sum of collagen and elastin contents of normal aorta showed progressive increasing with age, except for the groups younger than 20 years old, which showed a fall. But proportion of collagen to elastin concentration remained constant in wide age range in the aorta, though markedly diminished with advancing age in the pulmonary artery.
The susceptibility of aortic elastin to elastase was expressed as percent solubilized protein N of total elastin N, incubated with elastase in pH8.7 borate buffer under shaking at 37°C for 14 hours. These results showed progressively diminished values with advancing age, but there was no significant difference in the values between sclerotic area and adjacent normal area of the same aorta.
Activating by ultraviolet light, the fluorescent spectrum of the solubilized aortic elastin was drawed, using spectrophotofluorometer. Of three fluorescent substances recognised, one showed age-related increasing tendency after 40 years old, whose activation/fluorescence maxima was at 336/400nm. And the intensity of the fluorescence of aortic elastin was not influenced by sclerotic changes.
Elastase solubilized aortic elastin was chromatographed using Sephadex-G-100 column. All the samples of aortic elastin showed two peaks, Fraction A with larger molecular weight, and Fraction B with smaller molecular weight. The ratio of A/A+B showed increasing age trend, except for a case of 75 years old, which showed a fall. Elastin from sclerotic area showed higher A/A+B ratio than adjacent normal area of the same aorta.
As carbohydrate components of glycoproteins in the elastin fraction from the aorta, hexoses, hexosamines, methylpentose and sialic acid were analyzed. The proportion of the total sugar in elastin fraction showed age related increasig tendency. Of carbohydrate moiety in elastin, hexoses showed definitely increasing tendency with age, but reverse was true in hexosamines.
Higher total cholesterol and also ester cholesterol levels in elastin fraction from aorta were proved in sclerotic portion, comparing with adjacent normal portion.
From these results it was highly probable that these changes in elastin during aging and atherosclerosis might offer the conditions, attributable to atherogenesis.

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1. Acidic glycosaminoglycans (AGAG) in arterial tissue were reviewed biochemically. It was followed by general biochemical methods for the analyses of the aortic AGAG, combined with the results of differential analyses in the intima, media and adventitia in human and bovine aortae.
2. The aortic AGAG existed in higher contents in the inner layers than in outor layers. Gel filtration by Sephadex G-25 after digestion with chondroitinase-AC indicated that the concentration of chondroitin-4-and -6- sulfates is higher in the intima than in the adventitia.
3. Electrophoretic separation prior to and after enzymatic digestion of aortic AGAG indicated that the AGAG in the adventitia predominated the ratio of dermatan sulfate, heparan sulfate and hyaluronic acid.
4. These results were substantiated by the data obtained with enzymatic digestion at the disaccharide subunits: paper chromatographic separation followed by chemical analysis on the separated unsaturated disaccharides.
5. Physiological function of the aortic AGAG was manifested as anticoagulants by thrombelastography. Dermatan sulfate and heparan sulfate in the aortic AGAG exhibited the potent property among the other AGAG. It was suggested that not only AGAG but also proteoglycans as macromolecules in the vessel wall play a function as anticoagulants and antithrombogenic agents against the atherosclerotic process.

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In human as well as animal atherosclerosis, the bulk of lipids accumulates in lesions in the form of small spheres. We have studied these particulate lipids in human fatty streak and fibrous plaque lesions, and determined their average diameter as 1.9μ and defined their liquid crystalline nature as lyotropic smectic mesophase. We have designated these lipid bodies in atherosclerosis as lipid inclusions.
In this study we isolated these lipid inclusions from fatty streak and fibrous plaque lesions of aortic atherosclerosis of each 21 subjects, with an average age of 40.9 and 51.2, respectively, who died sudden and unexpected death. Lipids were analysed by quantitative thin-layer chromatography and cholesteryl ester fatty acids by gas-liquid chromatography for comparison between inclusions and inclusion-free tissue residues, and also between the inclusions of fatty streak and fibrous plaque lesions.
The compositions of lipids of the inclusions were remarkably uniform and strikingly different from those of inclusion-free residues, respectively: cholesteryl esters 82.3 vs 34.6% free cholesterol 4.9 vs 20.4%, triglycerides 4.3 vs 4.8%, and total phospholipids 8.7 vs 40.1% in fatty streaks, and cholesteryl esters 60.8 vs 42.0%, free cholesterol 13.0 vs 28.9%, triglycerides 10.4 vs 6.3% and total phospholipids 15.8 vs 22.9% in fibrous plaques. The proportions of individual phospholipids were also different in lecithin and sphingomyelin between the inclusions and the residues in fatty streaks. The compositions of inclusions of fatty streaks were significantly different from those of fibrous plaque inclusions with the exception of triglycerides and lysolecithin. Major fatty acid of cholesteryl esters in both fatty streak and fibrous plaque inclusions was oleate.
These results indicate that (i) the lipid inclusions from both fatty streaks and fibrous plaques are quite unique in the chemical compositions when compared with those of any other blood lipids or serum lipoproteins such as chylomicron, VLDL, LDL, and HDL. (ii) that the metabolic origin of the lipid inclusions, therefore, cannot be explained by a simple deposition of blood lipid constituents or serum lipoproteins in the lesion tissues, and (iii) that the liquid crystalline nature of the inclusions largly due to physical properties of cholesteryl ester molecules seems to be playing a crucial role in the pathogenesis and development of fatty streak and fibrous plaque lesions.

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Properties and characteristics of cholesterol ester hydrolase in the human aortic intima and media have been investigated.
1) The accumulation of cholesterol ester in the aortic wall might not be caused by the decrease of cholesterol ester hydrolase activity.
2) Cholesterol ester hydrolase in the human aortic intima and media had been purified 80-100-fold by gel filtration.
3) Two pH optima for the partially purified cholesterol ester hydrolase were observed at pH5.0 and 7.0-7.5.
4) With respect to the effect of substrate concentration on the enzyme activity, the acid cholesterol ester hydrolase was inhibited by the reaction products in high concentrations, and the neutral one by the emulsifiers.
5) Phospholipid, which was used with sodium taurocholate or albumin as a emulsifier for substrate, increased the activity of cholesterol ester hydrolase.
6) The present results suggested that there were some inhibitors for the activity of cholesterol ester hydrolase in the aortic wall.

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Blood fibrinolitic activity and plasma fibrinogen level was measured in 44 patients with arteriosclerosis obliterans, 6 patients with thromboangiitis obliterans and 40 patients with diabetes mellitus. Furthermore, blood fibrinolytic activity and plasma fibrinogen level was measured in 14 healthy control men and blood flow was measured in 10 healthy individuals.
Mean plasma fibrinogen level was 300.9±8.8mg/dl in the normal controls and it was significantly (p<0.01) elevated in the patients with arteriosclerosis obriterans (379.5±22.3mg./dl). Mean euglobulin lysis area on the standard plates was 107.0±5.1mm² in the normal controls and it showed to be significanty (p<0.01) decreased in the patients with thromboangiitis obliterans (67.0+14.4mm²) and in the diabetics (67.5±6.6mm²). The lysis area on the heated plates induced euglobulin plus streptokinase was 100.3±3.4mm² in the normal controls and it showed to be significantly increased in the patients with arteriosclerosis obliterans (113.6±2.4mm²), although it showed no significant differences among these four groups on the standard plats.
Post-ischaemic reactive hyperamia blood flow though the anterior tibial muscle was 21.7±SE1.2ml/100g/min in the normal controls. In patients with arteriosclesosis obliterans, blood flow was 11.8±1.8ml/100g/min the unaffected legs (p<0.01) and 10.0±0.7ml/100g/min in the affected legs (p<0.01). In patients with thromboangiitis obliterans, it was 13.3±1.4ml/100g/min in the unaffected legs (p<0.05) and 13.1±1.9ml/100g/min in the affected legs (p<0.01). Blood flow was 14.9±1.2ml/100g/min in the patients with diabetes mellitus (p<0.05).
Blood flow level was analysed against plasma fibrinogen level in the patients with arteriosclerosis obliterans and diabetes mellitus. In these diseases blood flow showed to be more decreased than normal level which was obtained from the follwing equation.
log(Blood Flow)
=-0.0006(Fibrinogen)+1.47
Therefore, it was suspected that decreased muscle blood in diabetics was due to the microangiopathy in the muscle, although decreased blood flow in the patients with arteriosclerosis obliterans was mainly due to the macroangiopathy.

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In many previous reports, evidences have been presented that plasma low density lipoprotein may be a major source of lipids in the naturally occurring atherosclerotic plaque. The well established Coon's fluorescient antibody technique has been employed to identify native tissue antigens in study of arterial lipoprotein.
Specific antibody against serum low density lipoprotein, labelled with f luorescin, has been used as a histochemical stain to demonstrate the presence of the lipoprotein in the arterial wall. Histological materials from normal and atheromatous aortas and large arteries has been obtained from the 1% cholesterol fed rabbits and the human subjects autopsied with 2-6 hours after the death.
Results obtained are as follows: No immunofluorescin of serum low density lipoprotein in the aortic wall were observed in the normal rabbits. On the contrary, in the cholesterol fed rabbits, fluorescin were observed within intima, media and adventitial tissues, indicating insulation of the serum lipoprotein from the lumen through the wall, then into the surrounding tissues. However, quantity of accumulated lipoprotein inside the arteries appeared in proportion to the extent and duration of hyperlipidemia.
In the all of the materials obtained from human subjects, fluorescin of the lipoprotein observed inside the arterial wall. And degree of the lipoprotein accumulation inside the aortic wall seems in proportion to the age of the subjects.
In the atheromatous section of human subjects, striking accumulation of the anti-lipoprotein fluorescin existed arround the surrounded tissues of the plaque.

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In order to elucidate the serum lipoprotein metabolism, ¹⁴C-1-glutamic acid and ³H-1, 2-cholesterol containing serum were intravenously injected to control, cholesterol-fed and aged rabbits. Lipoproteins were separated ultracentrifugally and radioactivities were counted by Aloka liquid-scintillation counter.
About 90% of ³H-cholesterol was incorporated into the lipoproteins and about 45% of ¹⁴C-glutamic acid was into apoproteins. Almost of incorporated ³H-cholesterol existed in the LDL, especially in the cholesterol-fed group in which radioactivity of ³H-cholesterol was remarkably high (50×10⁴ DPM/serum 1ml). In the control and cholesterol-fed groups, high levels of radioactivities in the LDL were continued during the time course. However, in the aged group, uptake of ³H-cholesterol (3×104 DPM) was less than those of above mentioned 2 groups and decreased faster. In the cholesterol-fed group, uptake of ³H-cholesterol into HDL lipid fraction was much lower than those in another groups.
Uptakes of ¹⁴C-glutamic acid into LDL apoproteins were almost consistent in the 3 groups. However, during the time course, radioactivities of apoproteins decresed in the control and aged groups, although the radioactivity continued in high level for 6 hours after administration in the cholesterol-fed group. Similar tendencies were observed in the uptake of ¹⁴C-glutamic acid into VLDL apoproteins and the uptake was almost 2 times higher than that into LDL.
From the above mentioned results, it may be concluded that, in lipoprotein metabolism, turnover of apoproteins were not so much affected by the age and cholesterol administration but also the turnover of lipid fraction were affected.
Farthermore, from the aspects of specific activities, cholesterol turnover of LDL and VLDL were rather depressed in the condition of cholesterol administration.

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