Endothelial cells were cultured from human thoracic aortas obtained at autopsy. Cultured endothelial cells from 27 out of more than 100 cases were examined as pure enough for the study by vWF immunostaining at the primary culture. Multinucleated endothelial cells (Variant endothelium) with vWF and Weibel-Palade body were mixed in the primary cultured cells from adult aortas. Frequency of concomitant variant endothelial cells among typical small cells correlated well with severity of atherosclerosis and partly with aging. The reason for the latter seemed to be the result of selective culturing from older persons with milder atherosclerosis.
Prostacyclin release from cultured endothelial cells was examined under three groups of young, middle, and old at pulsatile ambient pressures. It revealed that endothelial cells from younger persons had a higher capacity to produce prostacyclin and to react to changes in pulsatile pressures. Endothelial cells from middle and older persons had lower productivity of prostacyclin and lower reactivity to pressure changes.
We concluded that endothelial function and morphology gradually altered over the decades and their alteration was well correlated with atherosclerosis. However, it is not known whether the existance of variant endothelial cells is causative or resultant of atherosclerosis.

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Thrombomodulin (TM) is a newly described protein that functions as a potent natural anticoagulant by converting thrombin from procoagulant protease into an anticoagulant. TM is found on the endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. In the present study, we investigated the immunohistochemical localization of two endothelial markers, TM and von Willebrand factor (vWF) in sections were thrombus. The endothelial lining in sections of artery with thrombus was positively stained by vWF antibody, while the staining of TM was very weak or negative.
Exposure of cultured human umbilical vein endothelial cell (HUVEC) monolayers to thrombin (1nM) induced changes in the shape of HUVEC and decreased the cell-surface TM molecule number by approximately 50%. The shape-change resulted in formation of gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. This gap-formation by thrombin was reversible and inhibited by the addition of dibutyryl cyclic adenosine monophosphate. These results suggest that intravascular thrombin formation may induce morphologic and functional alteration in the endothelium. The alteration in the endothelial lining in the blood vessels may have some role in the pathogenesis of thrombosis.

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Angiogenesis related to atherosclerosis is observed in the atherosclerotic plaque itself and underlying media. Medial hypoxia resulted from intimal thickening might be one of the causes of angiogenesis in the media, because in vitro studies show that conditioned medium of bovine aortic smooth muscle cells cultured under hypoxic condition caused remarkable angiogenesis, i.e. tube formation of cultured capillary endothelial cells in the collagen gel. The length of the angiogenic tube stimulated by conditioned medium of smooth muscle cells cultured under 1% O₂ is about 3 times and 20 times longer than that of 5% O₂ and controls, respectively. When plasminogen was in the gel, tube formation accelerated. Plasmin inhibitors, such as Trasylol, epsilon-aminocaproic acid, soybean trypsin inhibitor, and α₂ plasmin inhibitor, reduced the length of the tube. Antiurokinase type plasminogen activator (anti-u-PA) also reduced the tube formation. Some investigators reported that endothelial cells produced and released procollagenase. These results suggest that one of the mechanisms involved in invasion of endothelial cells is related to the protease cascade on the endothelial cell surface, in which u-PA, plasmin and collagenase participate.

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Epicardial coronary artery diameter and coronary blood flow were measured by a sonomicrometer and electromagnetic flow probe, respectively, in conscious dogs. Reactive dilation appeared following the full appearance of reactive hyperemia. Serotonin induced coronary dilation as well as reactive dilation disappeared after endothelial balloon-denudation. Thus, endothelial linings play an important role in regulation of the epicardial coronary arterial tone in physiological states. Reduced level of endothelium-related relaxation in response to serotonin was also noted in Göttingen miniature pigs. When severe and persistent spasms were provoked in pigs, intramural hemorrhage was frequently induced along the thickened intima. The latter phenomenon suggests the important relevance of spasms in the progression of atherosclerosis or evolution of myocardial infarction.

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For the simple examination of the change in vascular permeability response of endothelial cells to various stimuli, we attempted to establish an in vitro experimental model using Boyden's chamber. This chamber has both upper and lower compartments with a partition filter between them. We succeeded in obtaining a monolayer of vascular endothelial cells on the surface of the filter by using a gelatin coated nucleopore filter. Both upper and lower compartments were filled with culture medium. When bovine serum albumin was added only to the upper compartment, the albumin was time-dependently leaked from the upper compartment to the lower compartment though the pores of the filter. The leakage was inhibited greatly when the surface of the filter was covered with entothelial cells.
By monitoring the amount of albumin leaked into the lower compartment after the addition of various stimuli, such as histamine, serotonin, bradykinin, PAF (Platelet activating factor) etc., to the upper compartment, we could judge which stimulus could better influence vascular permeability. PAF was found to be the most active stimulus in increasing vascular permeability.
During the course of the investigation, we found that such substances as having cytotoxic activity, i.e., lipid peroxides, antiserum in the presence of the complement etc., could also increase vascular permeability. Therefore, by measuring cell viability at the same time, this model can be used as an in vitro assay system for measuring vascular endothelial cell injury. This model may also be a useful in vitro assay system for the evaluation of cytoprotective drugs. By using this system we found that 15-HPETE (15-hydroxyeicosatetraenoic acid), one of the lipoxygenase metabolites of arachidonic acid, has a strong cytolitic activity on vascular endothelial cells and that MCI-186 (3-methyl-1-phenyl-2-pyrazolin-5-one) could prevent the cells from such injury.

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Changes in lipids in whole serum and serum lipoproteins were determined after a single injection of Triton WR-1339 into both splenectomized rabbits and control rabbits. 14 white Japanese male rabbits were divided into two groups: a splenectomy group (Spx group n=8), and a control group (Sham group n=6). After the operation, Triton WR-1339 at a dose of 200mg/kg body weight was administered to the rabbits. Lipids in whole serum and lipoproteins were measured at 0, 6, 24, 48, 72, and 120hr after injection.
Triton-induced hyperlipidemia occurred in both groups. However, we found significantly lower levels of serum triglyceride (TG) and TG in very low density lipoprotein (VLDL) at 6 hours after injection in the Spx group and also found no increase in TG to total cholesterol (TC) ratio in VLDL. These data suggest that an inhibitory effect of Triton on lipoprotein lipase activity was partially suppressed from 0 to 6hr in the Spx group.
The lipids in low density lipoprotein (LDL) fraction of the Spx group were higher than those of the Sham group at 120hr. This may indicate retarded LDL catabolism in the Spx group and may provide us with a possible explanation for our previous observation: more enhanced dietinduced hyperlipidemia in splenectomized rabbits.

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Patients with diabetes mellitus also frequently have atherosclerotic diseases such as myocardial infarction and/or stroke which exert a great influence on prognosis. We measured levels of thrombin-antithrombin III complex (TAT), crosslinked fibrin degradation products (XDP), tissuetype plasminogen activator (t-PA), and elastase-α₁ proteinase inhibitor complex (E-α₁ PI) in plasma in 20 diabetics. Levels of TAT, which is considered to be a sensitive marker of intervascular activation of prothrombin, and E-α₁ PI, which is thought to reflect the amounts of elastase derived from polymorphonuclear neutrophils (PMN) were found to increase significantly in diabetics compared to controls. Levels of t-PA, which converts plasminogen to plasmin, tended to decrease and concentrations of XDP were within normal limits in diabetics. The levels of E-α₁ PI in plasma of diabetics with high levels of TAT (5.0<) was lower than those with low levels of TAT, although this was not statistically significant. From these results, we conclude that hypercoagulability and impaired fibrinolysis are present in diabetics which enhance thrombotic tendency. The fact that concentrations of E-α₁ PI in plasma did not increase when levels of TAT increased indicates that host defense to development of atherosclerosis by PMN-elastase might be inhibited in diabetics with hypercoaglability.

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Antigenic material recognized by monoclonal antibody EMR1a/212D against extracellular matrix deposited with lipid was purified from serum of WHHL rabbits. The antigenic material was purified in a single band by SDS-PAGE followed by DEAF-Sepharose CL-6B column, EMR1a/212D coupled immunoaffinity column, and HPLC equipped with TSK gel G3000SW_XL, column chromatography. The purified antigenic material was a glycoprotein with a molecular weight of 66k daltons. The amino acid residue of the antigenic material was remarkably high in glutamic acid and aspartic acid. However, the residue was low in arginine and lysine. Its isoelectric point was at pH 5.4-5.9. The antigenic material contained 22.2mg sugar per 100mg protein, and at least sialic acid expressed the activity of the epitope, because antigenic activity was decreased by neuraminidase treatment.

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The role of protein kinase C pathway and Ca⁺⁺-calmodulin system in prostacyclin (PGI₂) generation in cultured human vascular endothelial cells (VEC) was investigated. VEC were isolated and cultured from human umbilical cord veins. PGI₂ released from VEC was assayed by RIA. Cytosolic free Ca⁺⁺ concentration ([Ca⁺⁺]_i) was measured using fura-2/AM. Results were as follows: The basal level of [Ca⁺⁺]_i in VEC was lower (7.27±1.41×10^-9M) than those of other cells. Released Ca⁺⁺ from storage sites was considered to be essential for the maintenance of basal PGI₂ generation. From the experimental results, the initial increase of [Ca⁺⁺]_i was necessary and the basal PGI₂ generation was also maintained in the Ca⁺⁺-free solution. The addition of Ca⁺⁺ ionophore A23187 (A23187) or bradykinin (BK) increased [Ca⁺⁺]_i and PGI₂ generation. The increase of [Ca⁺⁺]_i induced by A23187 was sequential while the increase induced by BK was transient. The pretreatment of the cells with a calmodulin inhibitor W-7 (10^-5M, 10min) or a protein kinase C inhibitor H-7 (10^-5M, 30min) suppressed the enhancement of [Ca⁺⁺]_i and PGI₂ generation induced by A23187. Although pretreatment abolished these increased parameters induced by BK. Through these results it was concluded that protein kinase C pathway and Ca⁺⁺-calmodulin systems contributed partially to increased [Ca⁺⁺]_i and PGI₂ generation induced by A23187 or BK.

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The effect of BF-759 (sustained release preparation of bezafibrate, 200mg/tablet, twice a day for 12 weeks) on serum lipids and apolipoproteins was investigated in 20 patients with hyperlipidemia. In 9 hypercholesterolemic subjects (hyper TC group), BF-759 caused average decreases of serum total cholesterol and LDL-C levels after 8 weeks by 14.8 and 24.2% respectively. In 11 hypertriglyceridemic subjects (hyper TG group), the drug decreased the triglyceride level after 8 weeks by 55.2%. HDL-C level increased by 36.1% in the hyper TC group, and by 39.2% in the hyper TG group. These changes were chiefly due to an increase of HDL₃-C. Apo A-I increased by 28.3, 17.7% in each group and apo A-II increased by 38.3, 30.2% as well. Apo B was reduced by 19.9% in the hyper TC group. Apo C-II, C-III, and E levels decreased in both groups. The atherogenic index also decreased significantly in both groups. Appreciable side effects were not observed during therapy. We concluded that BF-759 is a safe and effective serum lipid-lowering drug in patients with hypercholesterolemia and patients with hypertriglyceridemia.

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We examined the serum lipid levels and the serum apolipoprotein levels in 6 female patients (16-21 years), who were hospitalized due to anorexia nervosa in National Okayama Hospital 1986 through 1988.
The levels of serum total cholesterol of the six anorexia nervosa patients were higher than those of age-sex matched controls. The levels of LDL-cholesterol and apolipoprotein B were higher than controls in two cases. However, the levels of HDL-cholesterol and apolipoprotein A-I were higher than controls in the other three cases. In these three cases, however, apolipoprotein A-II and apolipoprotein C-II were lower than controls.
The levels of triglyceride of total cases were similar to those of controls.
These results suggest the existence of two kinds of hypercholesterolemia in anorexia nervosa, namely hyper LDL-cholesterolemia and hyper HDL-cholesterolemia.

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LDL apheresis is one of the promising procedures for treatment of severe familial hypercholesterolemia. In the present study, we developed a system for in vitro apheresis circuit to characterize and compare the function of two types of LDL apheresis modules: membrane filtration (module M) and adsorption units (module A). Module M (pore size: 0.04μm, total membrane area: 0.1m²) and module A with polyvinylalcohol gels (total volume: 20ml), originally designed for rabbits (Asahi Medical Co., Tokyo) were examined in the present study. Plasma obtained during double filtration plasmapheresis in a patient with familial hypercholesterolemia was used as pooled plasma. One unit of heparin/ml of plasma was added to pooled plasma. Mean concentrations of lipoprotein cholesterol (C) and protein in pooled plasma were as follows: total-C 577.5mg/dl (100%), VLDL-C 50.3mg/dl (8.7%), IDL-C 23.5mg/dl (4.1%), LDL-C 452.2mg/dl (78.3%), HDL-C 51.5mg/dl (8.9%) and protein 4.8g/dl.
Pooled plasma was perfused through each module under a constant flow rate (2ml/min). Samples were drawn from the module outlet every 5 or 10 minutes. Experiments using either module were performed twice. While module M was completely occluded within 20 minutes, module A continued for 60 minutes. Total flow volume was almost the same (70-80ml) in all experiments. Sieving coefficient (SC) of lipoprotein cholesterol and protein was calculated according to the following formula: SC=concentration of cholesterol (mg/dl) or protein (g/dl) in the sample obtained from module outlet/concentration of cholesterol or protein in pooled plasma. SC of LDL-C was lower than those for VLDL-C and IDL-C (0.02 vs. 0.22, 0.29) in module A, while SCs of VLDL-C, IDL-C and LDL-C were equally very low (near 0) in module M. SCs of HDL-C and protein were higher in module A than in module M. Total cumulative inflow amounts (TIA) and total removal amounts (TRA) of lipoprotein cholesterol and protein at every sampling time were calculated and compared. In the study using module M, the final ratios of TRA/TIA were 0.99, 1.0, 1.0, 0.997, 0.95 and 0.81 in total-C, VLDL-C, IDL-C, LDL-C, HDL-C and protein, respectively. Those ratios in the study using module A were 0.78, 0.64, 0.57, 0.82, 0.58 and 0.26, respectively. These results indicate that module A may have relatively better removal selectivity for LDL and allow more HDL and protein to pass through the column and module M can remove a greater amount of LDL in a shorter amount of time.

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In order to clarify the relationship between apolipoprotein H in the liver, we determined plasma apolipoprotein H levels in patients with chronic liver disease by SRID method. The relationships of lipoprotein lipid, major apolipoprotein levels, liver function test to apolipoprotein H, and histochemical confirmation of apolipoprotein H in the liver were also investigated. 32 patients with chronic liver disease were classified into two groups by clinical and chemical examination; one group (mild) consisted of 17 patients with chronic hepatitis or liver cirrhosis without hepatic failure, and the other group (severe), were 15 patients with liver cirrhosis with hepatic failure.
The mean plasma apolipoprotein H levels in mild and severe groups were 14.0±2.9, and 9.8±3.6mg/dl, respectively. These were lower than in healthy subjects (21.9±2.4mg/dl) (both of p<0.001), and mean plasma apolipoprotein H level in the severe group was significantly lower than that in the mild group (p<0.01). Apolipoprotein H levels showed positive correlations to total cholesterol, cholesterol ester, triglyceride, HDL₃-cholesterol, apolipoprotein A-II, and apolipoprotein B levels (r=0.492; p<0.01, r=0.407; p<0.05, r=0.608; p<0.001, r=0.418; p<0.02, r=0.442; p<0.02, r=0.517; p<0.01, respectively), which were reported previously as indicators of liver functions. In addition, apolipoprotein H levels correlated positively with lecithin-cholesterol acyltransferase activities (r=0.458; p<0.01) and correlated inversely with ICGR (15) values and γ-globulin/total protein ratio (%) (r=-0.509; p<0.01, r=-0.387; p<0.05). Histochemical examination of the liver, using anti apolipoprotein H serum, revealed that apolipoprotein H existed in the liver cells.
These results suggest that most apolipoprotein H may be synthesized and secreted by the liver and plasma apolipoprotein H levels may reflect the severity of hepatocellular damage.

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Total cholesterol levels (TC) reached maximum (199mg/dl) at the 6th decade and decreased with age in males. However, TC levels increased with age in females and exceeded those of males after 6th decade. TG levels reached maximum (140mg/dl) at the 5th decade and declined with age, but those of females increased with age and exceeded those of males at the 6th decade. TC levels of both sexes were equ

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Since high density lipoproteins (HDL) fractions contain albumin when determined from serum by conventional precipitation methods, phospholipids in HDL do not demonstrate the accurate level of HDL-phospholipids. In the present study, true phospholipid levels in HDL were estimated using high performance liquid chromatography (HPLC), and the levels were compared with the phospholipids in HDL determined by the precipitation method. Serum from 18 healthy subjects was tested. Under the HPLC method, after extracting HDL fraction, subfractions of HDL were separated by monitoring cholesterol and phospholipids. It was found that phospholipids in the albumin fraction contained 20.2±1.6% (mean±S.D.) of the total HDL-phospholipids. Albumin-phospholipids correlated inversely with HDL₂-phospholipids (r=-0.472, p<0.05), and with HDL₂-cholesterol (r=-0.507, p<0.05). No correlation was observed between serum total phospholipids and albuminphospholipids. A significant correlation was observed between HDL-phospholipids determined by the precipitation method and HDL fraction phospholipids determined by the HPLC (r=0.903, p<0.001). These results suggest that HDL-phospholipids determined by the precipitation method gives useful clinical data.

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Acute effect of high cholesterol diet on 3-hydroxy-3-methylglutaryl Coenzyme A reductase (HMG CoA reductase) activity in peripheral blood mononuclear cells (PBMCs) was investigated in 5 healthy adult males. Four hours after a high cholesterol diet (fresh cream+4 boiled egg yorks), HMG CoA reductase activity decreased (p<0.1) compared to ingestion of only fresh cream. Free cholesterol content in PBMCs increased (p<0.1) after high cholesterol diet. These results suggests that down regulation of HMG CoA reductase appeared by increasing free cholesterol content in PBMCs after a high cholesterol diet.

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This study was undertaken to examine the change of contractile responses to vasoactive agents by atherosclerosis in the rabbit aorta.
Twenty-two male Japanese albino rabbits were divided into three groups: animals in group C were fed a normal diet, rabbits in group H were fed with 1% cholesterol diet for 3 months, and group S animals were fed with 1% cholesterol diet for 4-6 months. Immediately after sacrifice by exsanguination, the thoracic aorta was carefully dissected, to avoid rubbing the endothelial cells, and cut into 4×25mm helical strips. The strips were suspended in Krebs-Ringer solution kept at 37°C, aerated with 95% O₂+5%CO₂, and placed in organ bath under 2gr. resting tension.
Isometric force induced by KCl, norepinephrine (NE), 5-hydroxytryptamine (5-HT), and histamine (His) was measured with a force displacement transducer. After contractile examination, the aorta was photographed to determine the extent of atherosclerotic lesions. The degree of the lesion was graded into four classes according to the area of atheromatous plaque. Group S was subdivided into four groups: group S1 (area of atherosclerosis. 0-25%), group S2 (26-50%), group S3 (51-75%), group S4 (76-100%). The relationship between vasocontractile responses and the extent of atherosclerosis in the aorta was investigated.
Maximum tension generated by KCl decreased in group S1 when compared with group C. There was no significant differences between groups C, H, S2, S3 and S4. Maximum tension developed by 5-HT in group H, S3 and S4 were the same level as in the group C, but those in groups S1 and S2 were significantly lower than those in group C. Maximum tension generated by His was higher in group H and lower in group S than in group C. There was no significant difference in maximum tension generated by NE between the six groups.
ED₅₀ (median effective concentration, as an index of sensitivity to vasoactive agents) to KCl was not found to be different between the six groups. ED₅₀ to 5-HT decreased according to the extension of atherosclerosis. ED₅₀ to His in the group S3, S4 was higher than in group C. This means that the aorta with severe atherosclerosis was more sensitive to 5-HT, and less sensitive to His.
V_max (maximum velocity of developed tension at the concentration of approximate ED₅₀) decreased significantly and T 1/2_max (time from initiation of tension developing to half maximum tension at ED₅₀) for the four agents was significantly prolonged in group S4.
These results suggest that the changes of vasocontractility in atherosclerotic rabbit aorta are induced by the alteration of drug-specific pathways such as receptors or calcium mobilization in cell membrane. This is because the change of vasoreactivites are different between the degree of atherosclerosis or vasoactive agents.
It is thought that the decrement of V_max and extension of T 1/2 in the atherosclerotic aorta resulted from the mechanical change in the aorta caused by atherosclerosis, such as intimal thickening or increased stiffness of the arterial matrix.

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Dai-saiko-toh (Tsumurajuntendo, Tokyo) is one of the traditional Chinese drugs which has been recently been gaining attention in Japan. Following administration, aortas and coronary arteries were obtained from spontaneously hypertensive rats (SHR) and studied for their effects as an anti-atherogenesity. Sixty SHR were divided into three groups; A group: Dai-saiko-toh administration (900mg/kg/day), B group: Saikokaryukotsu-borei-toh (900mg/kg/day), and C group: vehicle feeding. They were orally fed with one regimen for. 3, 6, 9, and 12 months, respectively, under an identical environment. Blood pressure and body weight were monitored weekly until sacrificed. After the administration of these drugs, the samples for pathomorphology were taken from predetermined sites of aortas, coronary arteries, etc.
There were no significant differences in body weight, blood pressure between the experimental groups (A or B) and the control group. But serum triglyceride in the experimental groups tended to be lower than in the control group. Microscopically, aortas had more general and more severe fibrous thickening of intimas than those of coronary and other arteries. In addition, aortic intimas were generally more thickened in the control group than in groups A and B. There were more gross scars and perivascular fibrosis in myocardium of the control group than in the experimental groups. Although the results obtained reveal that there are no significant differences in blood pressure and various serum lipid concentration between the experimental and control groups, there are differences in aortic intimal thickening at 9 and 12 months. Dai-saiko-toh administration has been reported to have an inhibition effect on collagen synthesis or triglyceride absorption. Subsequently the aortic intimal thickening in SHR is suppressed as compared to that of control group and may have anti-atherogenesity, especially in SHR aortas.

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The effects of prazosin on the clearance of Intralipid and very low density lipoprotein (VLDL) were investigated in Wistar rats. The clearance rate of Intralipid and VLDL were higher in the prazosin administration group than that in normal controls. The transfer of phospholipid from VLDL to HDL fraction was more accelerated in the prazosin administration group than in the normal controls. Incorporation of free fatty acid (FFA) derived from VLDL-triglyceride into arterial wall was enhanced by prazosin (23.7±18.8dpm/100mg rat in normal controls; 43.8±20dpm/100mg prot in prazosin administration group). β-oxidation of [¹⁴C]oleic acid in cultured smooth muscle cells was not inhibited by prazosin. Prazosin did not increase cholesteryl ester synthesis from FFA derived from VLDL. These results suggest that prazosin might improve serum lipid levels by enhancing the clearance of VLDL. Enhanced FFA supply to aorta by prazosin was not responsible for atheroma formation.

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