Yasaburo Oike

Evaluation of Oxidized Low-Density Lipoprotein and Large Molecular Size Low-Density Lipoproteins in Atherosclerosis

To study the roles of modified low-density lipoprotein (LDL) and the molecular size of LDL in atherogenesis, the following studies were carried out. Eight white male rabbits fed a standard oriental diet with 1% cholesterol were used to isolate LDL and to observe changes in the molecular size of LDL due to cholesterol feeding. The tissue LDLs in the aorta were analyzed to confirm the existence of modified LDL (namely, LDL with peroxidized cholesteryl ester) by thin-layer chromatography. In addition, plasma LDLs were isolated from 18 patients with myocardial infarction and 11 patients with angina pectoris to confirm the existence of LDL with peroxidized cholesteryl ester. Each LDL separated consisted of 3 fractions; namely, IDL (1.006-1.018), LDL1 (1.019-1.052) and LDL2 (1.053-1.063) by sequential ultracentrifugation. The molecular sizes of LDL were measured by a planimeter from electron microscopic photographs, with negative staining. The estimation of peroxidized cholesteryl linoleate in LDL was performed using our method. The modified LDLs with peroxidized cholesteryl ester were poorly estimated in the LDL separated from the plasma of cholesterol-fed rabbits and from the aorta extraction after 16 weeks of feeding. The peroxidized cholesteryl ester was clearly identified in the plasma LDLs of the patients with myocardial infarction and angina pectoris, and in whole extracts from human aortic atheroma, although it was not clearly identified in the tissue LDL fraction. The molecular sizes of LDL1 enlarged week by week with cholesterol feeding, but two fractions of IDL and LDL2 did not change in size. The infusion of cholesterol-rich LDL of large molecular size or LDL with peroxidized cholesteryl ester into the vessels led to fixation, on the surface of the arteries of many platelets, red cells, and white cells, and to marked irregularities in the endothelial folds. The evidence suggests that atheromas, formed in a short period in rabbits with cholesterol feeding, are caused mainly by the increase in LDL1 of large molecular size, and that foam cells, formed in human atheromas, are caused mainly by the production of modified LDL with peroxidized cholesteryl ester.

Acceleration of platelet aggregability due to modulation of native LDL.

The aim of this experiment was to clarify whether low density lipoprotein (LDL) causes an acceleration of platelet aggregability. Native LDL was separated into two fractions by filtered tap-water dialysis, namely, water-soluble LDL (WS-LDL) and non-water-soluble LDL. Although native LDL did not enhance the platelet aggregability, WS-LDL made it markedly increased. WS-LDL consisted of the lipid constituents that were not found in native LDL. Namely, in thin-layer chromatography of WS-LDL, an unknown spot between triolein and free fatty acid was clearly stained. This unknown spot in the WS-LDL was produced by the peroxidation of cholesteryl ester in native LDL. It was confirmed that the spot has the same Rf value as the peroxidate of cholesteryl linolate in thin-layer chromatography. If native LDL is modulated by divalent metal ions and oxygen in the fluids, LDL with biological activity such as an increase of platelet aggregability is produced.

A new approach to prevention and treatment of atherosclerosis by dyslipoproteinemia.

A number of papers’.’ have been published on the treatment and prevention of atherosclerosis. The main points of those papers were to reduce the concentrations of low density lipoprotein cholesterol (LDL-CH), apoprotein B (apo B), and lipid peroxide (LPO) or to increase that of high density lipoprotein cholesterol (HDL-CH). Furthermore, in several human and nonhuman primate experiment^^.^ it was shown that atherosclerotic lesions regressed by reducing the concentration of LDL-CH for an extended period of time. Recently, Steinberg’ and Kitah reported that the foramtion of atheroma was suppressed in WHHL rabbits by probucol treatment. They pointed out that the suppression of LPO was more important for the treatment and prevention of atherosclerosis than the reduction of LDL-CH. In this paper, new aspects for the treatment and prevention of atherosclerosis will be discussed from the viewpoint of the composition and physical character of LDL.

Comparison among lipid constituents in native LDL, ultra-water-soluble LDL, and vessel wall, and their significance in arteriosclerosis

The lipid constituents in native low-density lipoproteins (LDL), ultra-water-soluble LDL (UWS-LDL), and aortic intimal tissues were compared. These lipoproteins were obtained from healthy persons and patients with atherosclerotic diseases. Also, aortic intimas were separated from arterial walls obtained within 5 hr after the donors death. (1) From the native LDL, cholesterol esters (CE), triglycerides (TG), small amounts of free fatty cid (FFA), free cholesterol (CF), and phospholipids (PL) were demonstrable by iodine vapor on TLC, but from UWS-LDL the above lipids plus a new lipid (spot X) were observed between TG and FFA on TLC. And also, an unknown spot with the same Rf value as spot X was recognized on TLC of lipid extract from the atherosclerotic lesion, but not from the normal intima. (2) The production of spot X in UWS-LDL is probably related to the oxidation of lipids in native LDL. Also, the spot X in UWS-LDL and the spot X in the atherosclerotic lesions are probably related to the oxidation of CE in these lipids. (3) The existence of UWS-LDL is important to the initiation and probably the progression of atherosclerosis.

Sodium ionophore converts growth manner of vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVES Vasoconstrictor peptides such as endothelin (ET) cause hypertrophy of vascular smooth muscle cells (VSMC) in Wistar Kyoto rats (WKY) and hyperplasia in spontaneously hypertensive rats (SHR). They also induce an increase in Na+ concentration ([Na+]i) and activate protein kinase C (PKC) independently. Therefore, we tested the hypothesis that the increase in [Na+]i may be involved in the conversion of growth manner under activated PKC in SHR VSMC. METHODS AND RESULTS 10(-7) M phorbol ester (TPA) increased the diameter and protein content of VSMC from both strains under 18% serum conditions. Further addition of 10(-6) M gramicidin (Na+ ionophore) converted TPA-induced hypertrophy to hyperplasia, which was due to the quick transition from S to G2/M phase, only in SHR VSMC. Western blot analysis showed that serum- and TPA-induced tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was potentiated by 10(-6) M gramicidin in SHR. [Na+]i, which was measured by sodium-binding benzofuran isophthalate (SBFI), was increased about 35 mM by 10(-6) M gramicidin in both strains, but TPA did not affect basal [Na+]i and the gramicidin-induced increase in [Na+]i. CONCLUSIONS We conclude that sodium ionophore may convert hypertrophy to hyperplasia synergistically with activated PKC in SHR VSMC, possibly by MAP kinase phosphorylation.

Endothelial Cell Injuries by an Infusion of Various Low Density Lipoproteins into the Rabbit Auricular Vein

Three-month-old white rabbits weighing 3 kg were used for the study. LDL was separated by Havel’s method. ’ The rabbits were divided into four groups by the kinds of feeding, namely, group 1, standard food; group 2 , group 1 + 1% cholesterol; group 3, group 2 + 2% probucol; and group 4, group 2 + 10% soycream (soycream was made from soybeans). LDL which was separated after feeding with each food for 5 weeks, was infused into the rabbit auricular vein. The concentration of the infused LDL was diluted to normal serum cholesterol level, and 400 ml of this diluted LDL were infused into the vein at a rate of 2.2 mumin for 3 hours. Similarly, LDL separated from the rabbits of the four groups was diluted to the same concentration, and the same amount was infused. Cerebral basilar arteries were observed by scanning electron microscope and transmission electron microscope,* and the findings were compared among the four groups. Platelet aggregability was measured by Born’s m e t h ~ d . ~

Batroxobin Accelerates Lipid Accumulation of Peroxidized Low Density Lipoprotein in Mouse Peritoneal Macrophages

Mouse peritoneal macrophages were incubated in DMEM medium with batroxobin (DF-521) to determine the effect of batroxobin on the internalization of peroxidized low-density lipoprotein (pox-LDL) by transmission electron microscopy. Although the morphology of the mouse peritoneal macrophages after incubation with DMEM, normal LDL (n-LDL) and n-LDL plus batroxobin was similar to that of the cells before incubation, they exhibited numerous cytoplasmic lipid droplets after incubation with pox-LDL for 4 h. After addition of batroxobin to the medium containing pox-LDL, the production of lipid droplets in the mouse peritoneal macrophages was tremendously accelerated. Batroxobin accelerates the phagocytosis and degradation of pox-LDL by macrophages.

Transformation of Cultured Human Monocytes by Peroxidized Low-Density Lipoprotein

To study the roles of peroxidized low-density lipoprotein (pox-LDL) during the formation of foam cells from human monocytes, monocytes isolated from normal human blood were incubated with RPMI alone, normal low-density lipoprotein (n-LDL) and copper pox-LDL. The concentrations of total cholesterol (TCH) and lipid peroxide in the human monocyte medium did not change significantly after incubation with RPMI medium alone or n-LDL, but those of TCH decreased slightly after incubation with pox-LDL for 24 h. Triglyceride (TG) concentrations in the culture medium of human monocytes decreased after incubation with all above substances. Ultrastructural studies showed that the monocytes changed to macrophages after incubation with RPMI alone or n-LDL and to foam cells after incubation with pox-LDL for 48 h. We conclude that TGs may be metabolized for ATP production by human monocytes. The energy may play a role in the transformation of monocytes to macrophages, and pox-LDL can induce transformation of monocyte-derived macrophages to foam cells.

Eleven cases with chilaiditi's syndrome in stroke patients.

昭和50年1月より昭和62年3月までの13年間に当院へ入院した脳卒中患者2,588例中に11例のChilaiditi症候群が認められた.男性7例, 女性4例, 発症時平均66.4±6.6歳であった.発症率は0.43%であり, 一般の集団検診における発症率より高く, 本症候群と脳卒中との関連が示唆された.全例とも特有の症状はみられず, 定期的な胸部X線写真により発見された.消化管の嵌入は一過性の場合が多く, その部位は総て結腸であった.発症の背景として, 全例に片麻痺があり, 頭部CT像および脳波から高度の脳機能障害の存在が推察された.100gGTT正常例は2例のみであった.腹部の合併症は, 胆石症と十二指腸潰瘍があり, 抗コリン剤の使用は2例であった.脳卒中発症から本症候群発症まで平均24.2±10.8ヵ月が経過しており, 長期のリハビリテーション治療を必要とする症例が多かった.成因として, 高度の脳機能障害による横隔膜および腸管の運動異常, および日常生活動作の著明な低下による腸管運動の低下などが考えられた.