Takemichi Kanazawa

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.

Oxidized LDLs but not native LDLs augment Ba2+ currents through L-type Ca2+ channels of the A7r5 smooth muscle-derived cell line.

The whole-cell patch-clamp method was used on A7r5 smooth muscle-derived cell line, and Ba2+ currents through Ca2+ channels were recorded. The A7r5 cells showed voltage-dependent, long-lasting Ba2+ currents which were markedly inhibited by nifedipine (10 microM). The magnitude of the maximum Ba2+ current (IBa(max)) was augmented by an application of dbcAMP (1 mM), but not affected by TPA (80 nM). Noradrenaline (NA) at 100 microM caused an increase in the IBa(max) by 19.7% in the presence of phentolamine (10 microM). This effect was cancelled by Rp-cAMPs (10 microM). In the presence of propranolol (10 microM), NA tended to reduce the IBa(max). Application of Ox-LDLs at 100 microg protein/ml caused an increase in the IBa(max) by 15.7%, whereas native LDLs did not change the IBa(max). Rp-cAMPs was ineffective to the Ox-LDL action on the IBa(max). In the presence of Ox-LDLs, NA augmented the IBa(max) by 21.4% in the presence of phentolamine. These results suggest that Ox-LDLs activate L-type Ca2+ channels of A7r5 cells by a mechanism independent of cAMP/PKA signalling.

Role of Renomedullary Thromboxane A2 in Development of DOCA-Salt Hypertension

To clarify the role of renal thromboxane (TX)A2 in the development of deoxycorticosterone acetate (DOCA)-salt induced hypertension, relationship between systolic blood pressure and the synthesis of renal TXA2 was investigated in 18 rats without and with OKY-046 administration which suppressed the synthesis of renal TXA2. Systolic blood pressure was significantly higher in DOCA-salt group than in control and OKY groups. The synthesis of 6-keto-prostaglandin(PG)F1 alpha and TXB2 in both renal arteries and cortical slices were more enhanced in DOCA-salt and OKY groups than in control group, but there was no significant difference between DOCA-salt and OKY groups. In contrast, the synthesis of 6-keto-PGF1 alpha in renomedullary slices did not vary significantly among three groups, and that of TXB2 was more increased in DOCA-salt group than in control and OKY groups. The cumulative sodium retention was significantly greater in DOCA-salt group than in control group. Administration of OKY-046 reduced the cumulative sodium retention in DOCA-salt rats by 45% toward that of the control group. These results might suggest that the enhanced production of renomedullary TXB2 was important to the development of DOCA-salt induced hypertension.

Chemiluminescence of hemoglobin and identification of related compounds with the hemoglobin chemiluminescence in plasma.

A low level of chemilumnescence by hemoglobin (Hb) was detected in the reaction with H2O2 and hydrogen donors such as gallic acid and catechins. The photon intensity was affected by the ferric state of Hb (methemoglobin > oxyhemoglobin), and was roughly correlated with the radical‐scavenging potential of catechins. We hypothesized the reversible activation reaction of Hb as the chemiluminescence mechanism of the H2O2/gallic acid/Hb system. It is indicated that the oxidized‐Hb (Hb‐OOH) formation was a chemiluminescence‐rate–determining step and one‐electron reduction by a hydrogen donor of the compound‐I–type intermediate ([·XFeIV]= O) proved a chemiluminescence‐specificity–determining step. Spectral analysis showed that the photon emission from the H2O2/gallic acid/Hb system was produced without singlet oxygen generation. The concentration dependence of photon intensity suggests a high consumption ratio of H2O2 leading to protection from H2O2 toxicity. Albumin was defined as a hydrogen donor by the isolation of chemiluminescent substance in plasma using this chemiluminescence system.

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.

Peroxidized low-density lipoprotein with four kinds of hydroperoxidized cholesteryl linoleate estimated in plasma of young heavy smokers.

AIM To clarify the mechanisms of vascular complications due to heavy smoking, it was studied whether hydroperoxidized low-density lipoprotein (HPO-LDL) was estimated in plasma of young heavy smokers. METHOD Plasmas were separated from 15 young male students (HS-M) who smoked more than 30 cigarettes/day over 5 years, and from 10 nonsmoker students (NS-M) too. LDL was isolated by ultracentrifugation. HPO-cholesteryl linoleate (HPO-CL) was identified by thin-layer chromatography (TLC), and lipid peroxide was measured by Determiner LPO (Kyowa Medics Co., Ltd, Tokyo, Japan). The molecular structure of lipid peroxide in LDL was identified using GMS analysis, HPLC chromatography and 1H-NMR analysis. RESULTS (1) HPO-CL was clearly observed on the TLC in LDL lipids of HS-M but from NS-M it was barely found. (2) Lipid peroxide in LDL separated from HS-M consisted of HPO-CL and reduced HPO-CL. CONCLUSION Peroxidized LDL was estimated in plasma with young heavy smokers. Because of injurious reactions in vessels of peroxidized LDL, it is considered that heavy smoking is one of the risk factors for vascular complications in heavy smokers.

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 . ~