Atherosclerosis is principal contributor to the pathogenesis of myocardial and cerebral infarction, gangrene and loss of function in the extremities. The lesion result from an excessive inflammatoryfibrous proliferative response to various forms of insult to the endothelium and smooth muscle cell has been known as “response to injury hypothesis of atherosclerosis”. In recent years a large number of types of toxic injury and diseases has been linked with free radical-mediated disturbances. In many cases, implication has been that the free radical disturbance is causative in relation to the ensuing cell or tissue damage. LDL can be oxidatively modified via a free-radical mediated process. Oxidixzed LDL (ox-LDL) is a key component in endothelial injury. Once ox-LDL is formed, ox-LDL may directly injure the endothelium and play and initial role in the increased adherence and migration of monocytes and T-lymphocytes into the subendothelial space. The earliest recognizable lesion of atherosclerosis is the so-called fatty streak an aggregation of lipid-rich macrophages and T-lymphocytes within the innermost layer of the artery wall, the intima. As one of the cause of the endothelial injury, has been linked with free radical mediated alteration of low density lipoprotein (LDL), i. e., peroxidized LDL. Ox-LDL is a key compornent in endothelial injury which increased adherence of monocyte/macrophage and T-lymphocytes. The macrophages scavenge the ox-LDL and become large foam cells which accumulate in fatty streak. Lack of antioxidants participate ox-LDL formation and endothelial dysfunction. Ascorbic acid is most strong antioxidant in the tissue. The principle pathway of ascorbic acid oxidation and turnover is believed to involve the removal of two electron in succession, yielding firstly the ascorbate free radical (AFR) and then dehydroascorbate. AFR may be reduced by a micorosomal NADH dependent enzyme, monodehydro-L-ascorbate oxidase to ascorbate. Dehydroascorbate also reduced to ascorbate as shown in Fig. 1. Thus, ascorbic acid acts as redox or antioxidant as a biological role in hydrophobic state. We have reported ascorbic acid deficiency, scurvy induced not only endothelium injury of the capillary but also of the aorta in guinea pig as shown in Fig. 2. Scurvy also induced an increase in serum lipoproteins in guinea pig due to suppressed lipoprotein lipase activity and reduced cofactor for 7α-hydroxylase which is rate limiting enzyme in the process of cholesterol to bile acid metabolism. Marginal vitamin C deficiency for 8 weeks induced fatty streak without supplemental cholesterol feeding in guinea pig. Recently, scurvy prone osteo-dystrophic rat (ODS-rat) with lack of L-gulonolactone-α-oxidase was separated (Fig. 3). Kawai et al, reported that mutant cDNA of the rat has a single base mutation from guanosine (G) to adenine (A) at nucleotide 182, which mutation alters the 61st amino acid residue from cysteine to tyrosine. In the serum of the ODS-rat, we could not find apparent changes of serum lipids but found significant increase in oxidized lipid estimated as MDA (malon dialdehyde) and LPO (lipo-peroxide) as shown in Fig. 4. By electone microscopic observation of the ODS-rat aorta, we found several cells which might be macrophage or lymphocyte with vacuolar structure in the cytoplasma in subendothelial space (Fig. 5). The importance of ox-LDL in atherosclerosis was established through the use of the antioxidant, probucol, in studies of a genetic hyperlipidaemic rabbit as model of atherosclerosis. As a rosis. Numerous antioxidants have listed in Table 1. Vitamin C and E act synergistically, vitamin E act as the primary antioxidant and the resulting vitamin E radical then reacting with vatamin C to regenerate vitamin E. Still a question remains whether such an interaction between vitamin C and vatamin E radical can take place in biological systems. Niki revealed that vitamin C located in the aqueous region c

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Adseverin of gelsolin family was distributed in neuronal cells, endocrinal cells and blood cells with exocytic function, but not in mormal adult smooth muscle cells. In primary culture of smooth muscle from bovine carotid artery, we found that this protein had gradually been expressed, arriving at maximum in confluent state by immunoblotting method.And then, we examined the expression of this protein in cultured cells time-dependently by immunofluorescent method as comparing with other structural proteins.
On 1st day of cultured cell by enzymatic preparation without serum, adseverin was not expressed in all the cells with deformed globular feature, but smooth muscle type of alpha-actin was clearly expressed. On 4th day after addition of serum, adseverin was expressed at the surface membrane of all the globular cells. In this state bipolar cells appeared under the globular cells in certain place. These cells had neither adseverin nor alpha-actin. Once bipolar cells appeared, their growth rate was very fast their numbers were rapidly increasing. Adseverin and alpha-actin were still not expressed in these cells, but stress fibers were observed by rhodamine phalloidin. When cells were going on confluent state, accompanied by post-confluent state with hills and valleys structure, globular cells were gathered, and formed tight round structure on the top of the hill.
On the other hand, in tissue of carotid artery adseverin was not detected in smooth muscle at the media, but at the hypertrophic area of intima adseverin was observed in small numbers of the cell. From these results we would like to propose the idea that there is a kind of stem cell of undifferentiated smooth muscle in the hypertrophic area of intima, and that these cells with globular form could be divided, but their cell cycle was very slow. Soon after different type of cell, perhaps undifferentiated cell with bipolar feature was separated and its growing rate was very fast. We discovered the stem cell of undifferentiated smooth muscle in bovine carotid artery primary cell culture and adseverin is an excellent marker of the cell, which means that this stem cell produce actively some growth factors using adseverin for exocytic function.

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We recently demonstrated that murine macrophage growth is induced by oxidized low density lipoprotein (Ox-LDL). The present study was undertaken to elucidate the mechanisms for Ox-LDL-induced macrophage growth. Whereas the mitogenic activity of acetylated LDL (acetyl-LDL) for murine resident peritoneal macrophages was negligible, treatment of acetyl-LDL with phospholipase A2 led to an increase in lysophosphatidyl-choline (Lyso-PC), and a concomitant increase in its mitogenic effect, indicating an essential role of Lyso-PC in macrophage growth. The role of the scavenger receptor was also examined. Maleylated bovine serum albumin (M-BSA), an effective ligand for the scavenger receptor but a poor carrier of Lyso-PC, did not induce macrophage growth even in the presence of Lyso-PC whereas acetyl-LDL induced cell growth in the presence of Lyso-PC. Moreover, Ox-LDL induced macrophage growth was inhibited by 70% by the presence of M-BSA. M-BSA competitively inhibited the scavenger receptor-mediated endocytic uptake of Ox-LDL, thus reducing concomitant up-take of Lyso-PC, whereas non-specific direct transfer of Lyso-PC from Ox-LDL to cell surface was not affected. These results suggest that internalization of Lyso-PC through the scavenger receptor is a keystep to generate an intracellular signal for macrophage growth.

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Characteristics of atherosclerosis are intima thickening, accumulation of large amount of lipids, and formation of foam cells in arterial walls. To understand mechanisms by which atherosclerosis is developed, we prepared various kinds of monoclonal antibodies which recognized particular regions of atheroma. Recently we established anti-oxidized LDL monoclonal antibody (FOH 1a/DLH3) using homogenate of human atheromatous plaque as immunogen. In this study we characterized antigenic materials recognized by the FOH1a/DLH3 antibody. The antibody reacted with oxidized LDL but did not with either native, acetylated or malondialdehydetreated LDL. The antibody cross-reacted with oxidized HDL, suggesting that particular sequences of apo B are not essential for specificity of the antigen recognition by the antibody. Immunohistochemical analysis of thin paraffin-sections from human colonary arteries showed that the foam cells derived from macrophages in atherosclerotic lesion were stained. The epitope of this antibody was further characterized by a model antigen generating system.
When the total lipid fraction extracted from LDL was treated with the ferrous ion-induced peroxidation system, the reaction mixture was recognized by the antibody. Antigenic product (s) was produced from phosphatidylcholine (PC) but not form the other lipid classes. The antigenic product (s) was capable to form a complex with a polypeptide. Binding of the complex of oxidized PC and polypeptide to FOH1a/DLH3 was competed by oxidized PC that was generated in the absence of any polypeptide, suggesting that oxidized PC product (s) is the epitope of the monoclonal antibody. We conclude that oxidized phospholipids product (s) is the epitope of the monoclonal antibody which forms complexes with polypeptides, and that the antigenic materials are detected in foam cells in atherosclerotic lesions. Based on our findings, we will discuss roles of oxidized PC in development of atherosclerosis.

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Coronary spasm plays an important role in the pathogenesis of coronaty artery disease. Augmented vasoconstrictor response to the stimuli may be due to both hyperreactivity of vascular smooth muscles to specific agonists and reduced endothelium-dependent vasodilatation. To clarify the mechanisms of impaired endothelium-dependent relaxation (EDR) in atherosclerotic arteries, we investigated the effect of Ox-LDL on EDR. Ox-LDL but not native LDL inhibited EDR. Approximately 40% of phosphatidylcholine (PC) was converted to lysophosphatidylcholine (LPC) during oxidative modification of LDL. Fractionation of individual lipids revealed that LPC derived from ox-LDL inhibited EDR. Ox-LDL or LPC inhibited accumulation of IP₃ followed by the increase of intracellular Ca²⁺ concentration induced by agonists including bradykinin. We clarified that low concentration of ox-LDL (10μg protein/mL) or LPC (5μg/mL) upregulated cNOS mRNA and protein levels by using RNase protection assay and immunoblotting. This action of LPC was abolished with cycloheximide but not with staurosporine. Furthermore, in situ hybridization and immunohistochemistry revealed that the cNOS mRNA and protein expression was normally observed in the endothelial cells overlying aortic fatty streaks in WHHL rabbits. These findings suggest that loss of EDRF activity associated with atherosclerotis is not due to an alteration of endothelial cNOS expression. Atherosclerotic arteries exhibit a marked increase increase of contractile response to 5-HT. We measured simultaneously both isometric tension and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in fura 2-loaded aortic smooth muscle strips. Athrosclerotic arteries from WHHL rabbit exhibited increased contraction associated with increase of [Ca²⁺]_i in response to 5-HT. Ketanserin, 5-HT₂ receptor antagonist, failed to inhibit supersensitivity to 5-HT in WHHL rabbit aortas. Moreover, WHHL rabbit aortas exhibited an augmented response to 5-carboxamide tryptamine, a 5-HT₁ like receptor agonist. We clarified 5-HT_1D and 5-HT₂ receptors were present in human coronary arteries using RT-PCR method. From these results, 5-HT_1D receptor-mediated signaling pathway may be involved in the supersensitivity to 5-HT in atherosclerotic arteries. Therefore, endothelial dysfunction by ox-LDL and LPC, and augmented contactile response caused by abnormal 5-HT signaling pathways in smooth muscle cells may play an important role in the abnormal regulation of the vasomotor tone in atherosclerotic arteries.

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