Since Lewis tried to cultuer hepatic endothelium, several authors had reported the in vitro culture of animal endothelium. However, the successive culturing for long time was not performed except by Kitsukawa or Pomerat et al. The author tried to culture the rabbits aortic endothelium in vitro over a year. 0.2% trypsin solution was adopted to gather the endothelial cells from aortic wall. The cells were cultured by 10% fetal calf serum in Eagle medium, and the 8 times “cloning” were done to get a pure strain of the endothelium.
1. Cultured endotheliums were asteroid or short fusiform in shape at early stage and changed their shape gradually to fibroblast-like long fusiform types.
2. Fibroblasts were also cultured by the same method from rabbits aortic adventitia as the control for cultured endothelium.
3. Electron-microscopically, endothelial cells had terminal bars in junctional complex, pinocytotic vesicles and band-like microfilaments which were seen also in human endotheliums.
4. The results of C. P. E. test (cytopathic effect test) showed different natures between cultured endothelium and fibroblast (TABLE 1).
5. Enzymologically, the cultured endothelium showed marked fibrinolytic activitied which were estimated about 4-5 times greater than that of cultured fibroblast.

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Influences of ascorbic acid (vitamin C) deficiency on the aorta and serum lipids were studied. Guinea pigs weighing about 300 grams were made ascorbic acid deficiency by scorbutic diet feeding.
1. Alteration of the aortic endothelium by manifest ascorbic acid deficiency, scurvy were investigated with Hautchen preparation, electron microscopy and frozen surface preparation for fibrinolytic activity according Warren. Aortic endothelium displayed reduced silver stainability of the cement line and an increased incidence of nuclear changes as vacuolization, swelling, distortion and pyknosis in Hautchen preparation (Fig. 2). Electron microscopy revealed separation of the endothelial junction and reduction of cytoplasmic organelles, and also a depletion of subendothelial collagen (Fig. 3). These findings indicate that ascorbic acid deficiency induces not only capillary alteration but also injury of the aortic endothelium. Fibrinolytic activity of the endothelium as an index of endothelial function was not changed by ascorbic acid deficiency, but displayed a gradient which increased from the arch to the abdominal segment of the aorta (Table 1).
Regional difference of the endothelial fibrinolytic activity may contribute to local difference on distribution of atherosclerosis.
2. Influcence of ascorbic acid deficiency on serum lipids and the aorta were studied. Male guinea pigs were fed with scorbutic diet with or without 5% coconut oil in weight for two weeks. Content of serum lipids, lipoprotein lipase activity and morphorogical change of the aorta were investigated. Serum triglyceride and cholesterol ester were moderately increased in ascorbic acid deficient guinea pigs, and markedly to twice of the normal control in the ascorbic acid deficiency with coconut oil feeding. Depression of plasma lipoprotein lipase activity was observed by ascorbic acid deficiency of both with and without coconut oil feeding. Histological examination of the aorta revealed edematous swelling of the ground substance in the intima and media in the scorbutic, and early atheromatous lesions of accumulated foam cells in the intima of the ascorbic acid deficiency with high fat diet (Fig. 4).
These findings suggest that ascorbic acid deficiency induces alteration of the aortic endothelium and increase of serum lipids and altered vascular wall connective tissue metabolism and consequently follow atherosclerosis.

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Obesity constitutes a risk factor for the development of coronary heart disease (CHD). However, it is not fully investigated which facet (s) of obsity is responsible for the development of CHD, since obesity accompanies varieties of disorders including physical overloard of overweight to cardiovascular organs, high blood pressure, impaired pulmonary ventilation, and metabolic derangements of diabetes mellitus, hyperlipidemia and hyperuricemia.
To investigate the role of physical overload of overweight to cardiovascular system in the pathogenesis of CHD, we have collected 48 cases of myocardial infarction (MI) and 19 cases of latent coronary insufficiency (LCI). They were grouped into 4 subgroups according to presence and absence of hypercholesterolemia, hypertriglyceridemia and hypertension. The distribution of patients in the 4 groups were: 20% for MI and 14% for LCI in the group where all the three of serum cholesterol, triglyceride and blood pressure were abnormal, 30% and 57% in the group where at least the two of them were abnormal, 40% and 21% in the group where at least one of the three was abnormal, and finally 10% and 7% in the group where all the three were normal, respectively.
The frequency of ischemic heart disease was the lowest in the obese subjects but without any accompanying metabolic and circulatory disorders of hyperlipidemias and hypertension. This finding was also supported by the fact that the occurrence of hypercholesterolemia, hypertriglyceridemia and hypertension distributes almost uniformly in the range of 12 to 20% among the 4 groups of obese subjects made in the same way as with MI and LCI patients.
These imply that MI and LCI were induced more strongly by the metabolic and circulatory disorders of obesity rather than the long-term physical overload of overweight itself to cardiovascular organs. Therefore, when we have an obese patient accompnying metabolic or circulatory disturbances, we have to treat him with more cautions since he has a higher risk for the development of ischemic heart diseases.

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The determination and characteristics of postheparin lipolytic activities, particularly monoglyceride hydrolase and triglyceride lipase, which played the major role in the removal of neutral lipids from the general circulation, were investigated.
When the low concentration of Triton X-100 was used as emulsifier in the assay medium, it could be measured the high activity of monoglyceride hydrolase.
The pH optimum of monoglyceride hydrolase in post-heparin human plasma was 9.0. This enzyme yielded a linear rate of release of glycerol from Triton X-100-stabilized monoolein emulsions at least 90min at 37°C. Although lipoprotein lipase was inhibited with higher concentration of NaCl, monoglyceride hydrolase was slightly activated with it. About 40% of lipoprotein lipase activity was lost during the pre-incubation for 30min at 37°C. However, monoglyceride hydrolase activity was completely stable at 37°C.
In order to investigate the origin of post-heparin plasma monoglyceride hydrolase, slices from rat tissues were incubated with or without heparin. Results of these experiments suggested that the post-heparin monoglyceride hydrolase was mainly of liver origin.
In clinical studies, lipoprotein lipase and monoglyceride hydrolase activities of hyperlipemic patients, who were classified according to the criteria of WHO, were determined and compared to those of the young and aged controls.

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The objective of this study is to establish a method of lipoprotein structure analysis. A preparative polyacrylamide gel (PAG) block electrophoresis of serum lipoproteins was combined with either antigenic component analysis or lipoprotein lipid analysis to constitute a systematic study system. In this work, workability of the formulated system has been evaluated applying it to some clinical and experimental studies.
Lipoprotein PAG block fractions evoked immune mechanism in rabbits, and the mechanism was employed to determine antigenic structure of canine and human serum lipoproteins. Five antigenic components were identifiable in canine serum lipoproteins by our hands. Albuminlipoprotein contained one specific antigenic component which does not show cross reaction with antisera from any other lipoprotein species. Alpha-1 lipoprotein consisted of two antigenic components, and both of them are common with those of alpha-2 lipoprotein. Alpha-2 lipoprotein consisted of three components; two of them were common with the alpha-1, and another was common with beta lipoprotein. Beta lipoprotein had two components; one was common with the alpha-2, and another was specific to the beta itself. In renogenic hyperlipoproteinemia, unusual lipoproteins were revealed frequently by PAG block electrophoresis, though they were not detectable on paper electrophoretograms. Several abnormal lipoproteins were identified through antigenic structure analysis, though total antigenic component analysis has not been completed in this study period. For example, an antigenic component other than ordinal apolipoprotein B component was detectable in the beta lipoprotein from a young-femals patient with advanced renal lesion. From methodological view point, these results seem to prove conclusively that lipoproteins are separated as structural-functional units effectively. As evidenced in this work, PAG block electrophoresis of serum lipoprotein combined with immunological techniques will be effective in characterizing unusual lipoproteins with which we may encounter. Lipoprotein lipid analysis was proved also to be an effective analytical method to characterize lipoproteins, especially as combined with other methods. Presently workable procedure by our hands consists of lipid extraction from lipoproteins separated with PAG block electrophoresis with chloroformmethanol, partial purification of extracts, thinlayer chromatography, detection of lipids on thin-layer plate with phosphomolybdic acid, and densitometric scanning of the chromatogram. Loss of the lipid moiety during electrophoresis is estimated as not more than those during manipulation after electrophoresis.

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In order to know abnormalities of blood coagulation in atherosclerosis and diabetes mellitus, relationship between plasma fibrinogen as factor I of blood coagulation and atherosclerosis was studied. As a result, plasma fibrinogen was found to have higher level in atherosclerosis than normal. And significant increase of plasma fibrinogen was shown in diabetics with atherosclerotic vascular complication, hypertensive patients with ischemic ECG tracing and cerebral stroke. Plasma fibrinogen in cerebral haemorrhage was shown to be increasing as compared with cerebral infarction. Plasma fibrinogen remained high untill three weeks after myocardial infarction. But no change of serum lipids was observed after attack. Therefore plasma fibrinogen was significantly increased in patients who had clinical manifestations of atherosclerosis. Also there was a close relationship between increasing fibrinogen level and patient's clinical course. These patient's atheromatous lesions in arterial wall were suspected to be progressive. If hypercoagulable state induced by the increase of fibrinogen often occures in atherosclerotic patients, thrombus formation on the atheromatous lesions will be accelerated resulting in arterial vascular occulusion. Then, ischemia and necrosis occuring in the peripheral vital organ will contribute to form clinical manifestations as infarction, haemorrhage and stroke. Whenever estimation of the course and prognosis in atherosclerotic patients is made, the follow-up fibrinogen measurement seems to be more necessary than serum lipids measurements such as cholesterol. It can be concluded that the increase of plasma fibrinogen in patients with atherosclerosis shows to reflect clinical manifestations caused by “thrombus formation” on atheromatous lesions. And it was found that there was a reverse correlation between lipolytic activity and fibrinogen concentration in diabetics and atherosclerotic patients.

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In this study has been described the potential for Elastase, an enzyme present in pancreatic juice, which participates in the decomposition of mucopolysaccharide or elastin to improve at least two factors associated with the developement of arteriosclerosis, i. e., metabolism of the arterial wall and metabolism of lipids.
It should be pointed out that any discussion of the influence of serum lipids on arteriosclerosis is not complete, if it dose not take into account a different effect of its carrier, lipoprotein, on the developement of this disease. With this consideration in mind, the influence of Elastase on plasma lipoprotein metabolism in the rabbits with normolipemia was investigated by the double isotope method in the present study.
Elastase (Eisai Co., Ltd.) was administrated orally to rabbits in a dose of 1050 Elastase units for 7 consecutive days. Ten days before administration of Elastase, 15μCi of ³H-1, 2-cholesterol was administered intravenously, and after 7 days of administration of Elastase, 70μCi of ¹⁴C-2-glycine was administered intravenously 6 hours before blood collection. And the blood levels of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) were determined by ultracentrifugation of blood samples.
When the influence of Elastase is viewed from the standpoint of the ratio of lipid to protein in each lipoprotein, the ratio of triglyceride to protein in VLDL is significantly higher (p<0.05), and the ratio of cholesterol to protein in LDL is also higher than in the controls. On the other hand, the specific activity of ³H-cholesterol in VLDL is lower in experimental animals than in the control, and there is no difference in its activity in LDL between the experimental and control animals. Similarly the incorporation of ¹⁴C-glycine into lipoproteins shows a lower value for VLDL-protein in the experimental animals, and presents no difference for LDL-protein between the experimental and control animals.
These facts, especially result in a high rate of metabolic turnover inspite of an increase in triglyceride against protein-unit in VLDL, suggest that the transformation of VLDL into LDL was promoted by Elastase. The increased ratio of cholesterol to triglyceride in LDL might serve as an indication that the decomposition of VLDL-triglyceride or early course of transformation of VLDL into LDL was pronoted by Elastase. Conversely, the decreased ratio of VLDL to LDL in ¹⁴C-protein would support the above view.
Importance is attached to disorders of lipoprotein metabolism, especially ones associated with various enzymes as one of the causes of hyperlipemia. In the light of the fact that Elastase exerted the above-mentioned effect in normal serum lipids, it can be expected that Elastase not only improves but also arrests hyperlipemia.

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