This study was designed to invertigate the mechanism of atherogenesis with reference to damage in the membrane system of endothelium in cholesterol-fed rats by using electron histo-chemical and immunohistochemical methods. The endothelial surface and pinocytotic vesicles revealed the existence of a ruthenium red-positive substance and colloidal iron was oberved at the anionic sites. The membrane bound enzymes such as Na⁺, K⁺-ATPase, were mostly located in the plasma membrane and pinocytotic vesicles of the endothelium. The administration of cholesterol resulted in the clumping and dispersion of ruthenium red and colloidal iron positive substances in endothelial cells, as well as the irregular distribution of Na⁺, K⁺-ATPase.
The membrane dysfunction caused by these molecular changes has been proved by the membrane permeability alteration as well as by the irregular distribution of the tracers, horseradish peroxidase and albumin. The authors wish to emphasize that endothelial damage may contribute to the increased permeability of plasma protein and lipids in the vascular wall, which may enhance atherosclerosis.

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We evaluated the effect of a low pathophysiological level of hyperlipidemia as well as the effect of in vitro exposure to LDL on the vascular responsiveness of isolated porcine coronary arteries. First, we fed pigs a cholesterol-rich diet for 4 weeks (C4 pigs) and 9 weeks (C9 pigs). The serum cholesterol level in the Cg pigs reached 218.5±32.9mg/dl compared with 85.5±8.4mg/dl in the controls. The Endothelium-dependent relaxation (EDR) caused by bradykinin, substance P, and serotonin were significantly reduced in the C9 pigs, but the relaxation induced by the Ca²⁺ ionophore, A23187 or nitroglycerin (NG) did not alter. Second, the direct effect of LDL in the presence of indomethacin was studied. The preincubation of LDL inhibited the EDR caused by bradykinin and A23187 especially in cholesterol-fed arteries, and also inhibited the EDR induced by substance P or serotonin in only cholesterol-fed arteries, not the control ones. The LDL did not affect the relaxation induced by the NG. These results suggest that EDR is attenuated by hyperlipidemia, and that the LDL directly attenuated the EDR especially in cholesterol-fed coronary arteries.

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In cultured bovine pulmonary endothelial cells (EC), human high density lipoproteins (HDL) induced DNA synthesis, which was measured by the incorporation of ³H-thymidine, in the complete absence of the serum. Neither the apolipoprotein components of HDL, apoHDL/cholesterol/DMPC (dimyristoyl phosphatidylcholine) vesicles, not the 25OH-cholesterol stimulated DNA synthesis. Co-incubation with phorbol-12-myristate-l3-acetate (PMA) did not affect the HDL-stimulated DNA synthesis. Diltiazem, one of the calcium channel blockers, inhibited DNA synthesis both in the quiescent and HDL-stimulated EC. The ⁴⁵Ca²⁺ uptake was not changed by incubation with HDL. These results suggest that HDL-stimulated DNA synthesis was not directly linked to the activation of protein kinase C or the extracellular Ca³ uptake.
The d<1.019g/cm³ fraction and LDL-stimulated DNA synthesis, in the presence of excessive amounts of these or IntralipidsR, did not cause any additional stimulation of DNA synthesis by HDL. Furthermore, oleic acid enhanced DNA synthesis in the quiscent EC, but not in the HDL (1mg prot/ml)-stimulated EC, thereby suggesting that lipoproteins may provide the fatty acids necessary for the proliferation of bovine vascular endothelial cells.

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The endothelial replacement of damaged endothelium was examined in the rabbit's thoracic aorta and carotid artery.
Endothelial cells, which line the arterial lumen in a monolayer, play important roles in permeability and phagocytosis by being selective during the influx of macromolecules and carbon particles, and with their functional motility and contractility in response to various stimuli. Endothelial cells have two unique properties: they resemble leucocytes and they contain microfilaments similar to those found in smooth muscle cells. Endothelial and smooth muscle cells display cell to cell communication, whereby regions of these cell types communicate by fusing together to form a junctional zone.
The rapid covering of a damaged endothelial area by viable cells (neighboring endothelial and smooth muscle cells) is important for preserving the endothelial layer. This phenomenon, observed under both adrenergic stimuli and hyperlipidemia, suggests that the endothelial dysfunction may be induced by various atherosclerosis risk factors:increased endothelial cell damage, increased endothelial cell death and replication, and structural or functional modification of the endothelium.
1. Endothelial damage (dysfunction) stimulates adjacent endothelial cells to either grow into large endothelial cells or prolong their marginal folds, which in turn cover the damaged endothelial cells.
The first type of protection (or replacement) can be observed in cases of prolongation of the marginal fold and large endothelial cells.
2. The second type of protection can be observed in cases of contraction. Activated large endothelial cells facing each other across a contracted eventually intrude into the valley.
3. The third type of protection occurs in cases showing a loss of continuity in the monolayer of endothelial cells;leucocytes then become trapped in the resulting gap in the layer. The trapped leucocytes in turn retain the monolayer continuity.
4. The fourth type of protection can be observed in cases of replacement with activated smooth muscle cells. In many of these cases, the endothelium shows extreme denudation or total damage. During this process, several mitogenic factors can induce smooth muscle cell activation and migration. Smooth muscle cell migration and proliferation occurs in atheromatous lesions and is promoted by the growth factors released by the macrophages.
These results support the contention that the various phenomena following the endothelial damage are the acute events related to the initial process of endothelial protection. The late migration of smooth muscle cells, on the other hand, may be responsible for initiating atherosclerosis.
Damaged endothelial cells are absorbed into the subendothelial space or carried away in the blood flow. After the endothelial replacement has been completed, smooth muscle cells and endothelial cells stop developing and the endothelium recovers a nearly normal appearance.

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Recent reports have suggested the important roles cyclic nucleotides play in endothelial physiology, including its selective permeability, its antithrombotic properties and its effect in regulating the vascular tone. However, cyclic nucleotide metabolism in vascular endothelial cells (EC) has not been extensively studied. We compared the endothelial adenylate cyclase, guanylate cyclase and phosphodiesterase (PDE) activities in the bovine aorta with the activities of the smooth muscle cells (SMC) from the same specimen in order to characterize endothelial cyclic nucleotide metabolism. We also investigated the possible role of cyclic nucleotides in the endothelial injury caused by activated platelets.
The adenylate cyclase and guanylate cyclase (soluble and particulate) activities of the EC and SMC varied greatly with respect to hormone responsiveness. The adenylate cyclase of the EC responded dramatically to the catecholamines, but did not respond well to prostaglandins (PGE₁, PGI₂) and glucagon. Meanwhile, the adenylate cyclase of the SMC responded to all of the above agents, although it responded less dramatically to the catecholamines. The soluble guanylate cyclase of the EC, unlike that of the SMC, did not respond well to sodium nitroprusside. The particulate form of the enzyme, however, showed a remarkable response to the ANP in the EC.
The PDE of the EC exhibited marked differences in the substrate specificity and isozyme pattern from the PDE of the SMC. The PDE activities of the EC homogenate were more potent in hydrolyzing cyclic AMP than in hydrolyzing cyclic GMP, while the reverse was true in the case of the SMC homogenate. The DEAE-cellulose chromatography of the soluble EC PDE revealed three isoforms; cyclic GMP PDE, cyclic GMP-stimulated PDE and cyclic GMP-insensitive cyclic AMP PDE. The calmodulin-stimulated form which was present in the SMC was not detected in the EC.
Human platelets, activated by collagen or lysed by sonication, enhanced the leakage of radio-activity from EC preloaded with [³H]adenine ([³H]adenine release). An analysis of radioactive materials by TLC revealed an intracellular decrease of the ATP and an adenylate energy charge and extracellular increase of the adenosine. The [³H] cyclic AMP showed time-dependent decrease in the prelabelled cells exposed to the activated platelets. Moreover, agents elevating the cyclic AMP levels prevented a [³H]adenine release.
We conclude that the EC possesses a cyclic nucleotide metabolism quite different from the SMC and that the cyclic nucleotides may play a role in protecting EC from agents which can potentially cause injury, including activated platelets.

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Effects of dietary monosaccharides on lipid metabolism were studied in rats with diabetes mellitus induced by streptozotocin (50mg/kg body weight). Fructose or glucose was supplied as 10 drinking water for 14 days. Diabetic rats showed an increased plasma cholesterol level, which was not affected by fructose or glucose. Diabetic rats receiving no sugar water had decreased rate of triglyceride secretion as compared to non-diabetic control rats. Whereas, plasma triglyceride concentrations were similar in the two groups, suggesting an impairment of triglyceride removal in streptzotocin-diabetic rats. Glucose feeding in diabetic rats produced no significant changes in plasma triglyceride concentration and triglyceride secretion rats. In contrast, fructose ingestion produced significant increase in plasma triglyceride levels in diabetic rates. This occurred in spite of the fact that dietary fructose resulted in no increase in the rate of triglyceride secretion. These results suggest that dietary fructose further interferes with plasma triglyceride removal in diabetic rats. Thus, the present data indicate that diabetes impairs triglyceride removal from the circulation and this impairment is further deteriorated by dietary fructose.

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The effect of the β-adrenergic stimulant (β₁ selective:denopamine, β₂ selective:terbutaline) on the serum lipid levels of a rat were examined. The drugs were administered at a dose of 10mg/kg over a 4 week period. The terbutaline group showed higher total cholesterol levels with both a, normal diet and a 1% cholesterol diet that did those of the denopamine group. The terbutaline group showed higher VLDL and LDL cholesterol levels with a normal diet than did those of the denopamine group. Free fatty acid and free glycerol levels were significantly lower in denopamine group with a cholesterol diet than it was in those of the control group.

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The cholesterol synthesis-suppressing activity of simvastatin was investigated to determine its clinical efficacy and effects on steroid hormones in type II hyperlipidemic patients. The patients were seven outpatients, and all were postmenopausal females with a mean age of 69.9 years (60-82 years). Simvastatin was administered once every day for 12 months. The daily dosage was 2.5mg in the first 6 months, 5.0mg in the following 4 months and 2.5mg in the last 2 months. The preadministration total cholesterol level in their plasma was 254.4±8.8mg/dl (mean±S. E.). During the 2.5mg administration periods, the total cholesterol and LDL-cholesterol decreased by 13.9±2.7% and 23.4±3.4% (p<0.01), while they decreased by 20.3±3.1% and 29.1±3.9% (p<0.01), respectively, in the 5.0mg administration period. These decreases were dependent on the simvastatin dosage (p<0.01). The HDL-cholesterol level did not change, the HDL-phospholipids level tended to increase, and the HDL-triglycerides level showed a clear increase. The ratio of the LDL-cholesterol to HDL-cholesterol decreased. The apo A-I and apo A-II increased in the early stages of simvastatin administration. Both the apo B and apo E decreased. From among the steroid hormones, cortisol, testosterone, estrediol, progesterone and aldosterone were measured. Simvastatin did not exert any influence on those steroid hormones, except aldosterone, which decreased significantly from the 9th month of administration. The aldosterone level decreased by 31.8±9.0% (p<0.05), from a preadministration level of 111.6±12.0pg/ml to 76.3±14.4pg/ml after 12 months of administration. No changes were detected in the ACTH level or in the plasma renin activity. Although there were no adverse reactions, the diastolic blood pressure slightly, but significantly decreased. This decrease in diastolic pressure dependended on the simvastatin dosage. These results indicate that simvastatin should be clinically effective in treating hypercholesterolemia. The increase in the HDL-triglycerides level suggests that HDL-cholesterol had been reverse-transferred to LDL. Although the observed decreases in the aldosterone level and the blood pressure are desirable phenomena, it is necessary to elucidate the long-term effects of simvastatin on steroid hormones.

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The metabolic changes of a living body are regulated by many factors, so it is important to understand the overall informations. The arterio-sclerosis is characterized by fibrous changes, accumulation of lipids and calcium on the arterial wall. The purpose of the present study is to examine many factors in the artery by multivariate analyses and to clear the changes in arteriosclerosis and the arterial components, relating to the etiology.
Aortae obtained from the 178 cases (124 men and 54 women) were examined by histochemical and biochemical methods, and the following results were observed.
1) The correlation between the content of the aortic wall components and aging were examined. a) Cholesterol of elastin fraction and the occupied areas by atheroma or calcification increased rapidly at a certain age. b) Both the medial elastin contents and the “polarity” of amino acid residues of elastin fractions in the media and the intima of the artery were kept continuously changing from young to old age. The qualitative and quantitative changes of the arterial elastin fraction occured in the arteriosclerotic artery.
2) The amino acids residues of elastin fractions were evaluated by multivariate analyses. a) Each 16 factors of amino acid residues in the intima and the media were studied by principal component analyses. As the results, the non-polar amino acid—glycine, alanine, etc.—residues were shown negative values of eigenvector in the first principal. On the other hand, the polar amino acid—glutamic acid, arginine, etc.—residues were shown positive values. The sample scores of the first principal component indicated total change of amino acid residues of elastin fraction, showing a positive correlation with the intimal and the medial polarity. It was supposed that the polarity implicated to indicate total changing of elastin fraction amino acid residues. b) The 32 factors of the intimal and the medial elastin fractions were examined by principal component analyses. As the typical results, such obvious difference was found between the changing in polar amino acid residues and non-polar ones by the first principal component, and it was true between the changing in the intimal amino acid residues of elastin fraction and the changing in the medial ones.
3) The 12 factors were studied by principal component analyses;that is, the intimal and the medial elastin contents, ealstin-cholesterol contents, polarity, desmosine and isodesmosine contents and atheroma and calcification. The following conclusions were obtained.
As a result of the eigenvectors of the first and second principal component, it was estimated that each 5 factors of the intimal and the medial aortic layers showed similar change, and it may be suggested that 12 factors changed sequentially and probably pathogenically in the order of the elastin content, cross-linked amino acids, polar amino acids, elasin-cholesterol and atheroma or calcification. The multivariate analyses allow the estimation and correlation of the various informations obtained from the living body in relation to diseases.

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The serum cholestanol level and the cholestanol/cholesterol ratio were found to be elevated in patients with heterozygous familial hypercholesterolemia, primary biliary cirrhosis, hypothyroidism and in cerebrotendinous xanthomatosis. These biochemical parameters were markedly increased in cerebrotendinous xanthomatosis. These two parameters, the serum cholestanol level and the cholestanol/cholesterol ratio, showed no significant difference of discrimination on diagnosing cerebrotendinous xanthomatosis. In cerebrotendinous xanthomatosis there was a significant positive correlation between the thickness of xanthomas of the Achilles tendon and the serum cholestanol level or the cholestanol/cholesterol ratio. Serum cholestanol elevation was not specific to cerebrotendinous xanthomatosis, however, the serum cholestanol level and the cholestanol/cholesterol ratio together appeared to reflect the severity of this disorder. All patients with CTX showed normocholesterolemia. The finding of both hypercholestanolemia and normocholesterolemia appears to be highly specific for diagnosing CTX.

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