Study for the mechanism of LDL modification search for its inhibitor.

动脉粥样硬化早期病变的特征之一是富含脂质的泡沫细胞积聚。最近的研究表明，大多数泡沫细胞来自单核巨噬细胞。这些巨噬细胞在细胞质中积累脂质，其中大部分来自低密度脂蛋白(LDL)。由于这些细胞表达的经典低密度脂蛋白受体数量很少，因此低密度脂蛋白掺入巨噬细胞的机制一直是广泛研究的焦点。报道称，通过乙酰化修饰的低密度脂蛋白可以被巨噬细胞上的一种受体识别和结合，他们将其命名为乙酰-低密度脂蛋白受体。然而，低密度脂蛋白的这种乙酰化似乎不太可能在体内发生。因此，一些研究人员一直在研究体内低密度脂蛋白的修饰形式。内皮细胞修饰的低密度脂蛋白、氧化修饰的低密度脂蛋白和香烟烟雾修饰的低密度脂蛋白被认为是天然修饰低密度脂蛋白的候选对象。和…近年来有大量证据表明，内皮细胞修饰的低密度脂蛋白与氧化低密度脂蛋白是一样的。Parthasarathy在体外观察到，通过化学氧化将低密度脂蛋白氧化成带负电荷的形式，使巨噬细胞转化为泡沫细胞，提示巨噬细胞摄取这种氧化的低密度脂蛋白的机制是由乙酰化低密度脂蛋白受体介导的。然而，在我们之前的研究中，我们发现乙酰化低密度脂蛋白对巨噬细胞氧化的低密度脂蛋白的降解仅有55%的抑制作用。这一结果使我们假设这两种脂蛋白的摄取机制可能不同。本研究通过比较氧化低密度脂蛋白、香烟烟雾修饰低密度脂蛋白和乙酰低密度脂蛋白与巨噬细胞的结合特性来验证上述假说。为此，我们在巨噬细胞的结合和降解方面进行了竞争和交叉竞争。结果清楚地表明，修饰低密度脂蛋白的巨噬细胞受体至少由三种不同的受体组成，它们具有不同的特异性。其中两个是这三种低密度脂蛋白的特异性受体，其余的是氧化和香烟烟雾修饰的低密度脂蛋白以及乙酰-低密度脂蛋白的共同受体。在用铜、铁或香烟提取物修饰低密度脂蛋白的过程中，我们发现超氧阴离子(O_2)起着重要的修饰作用，而超氧化物歧化酶(SOD)对这些反应有抑制作用。但过氧化氢酶不能阻止低密度脂蛋白的修饰。因此，我们筛选出了普罗布考、维生素E或维生素C衍生物等几种抗氧化剂来替代S.O.D。普罗布考是目前唯一能防止低密度脂蛋白修饰的抗氧化剂。其他试剂无法阻止这些反应，因为那些不能被包含在低密度脂蛋白颗粒中。接下来，我们利用WHHL-兔进行了体内实验，以了解普罗布考作为抗氧化剂的抗动脉粥样硬化活性。2月龄起饲喂1%普罗布考饲料17个月后，动脉粥样硬化斑块面积为23%，而对照组为93%。即使在8月龄时喂食1%普罗布考饲料，病变面积为54%，但6个月后病变面积平均约为38%。提示氧化型低密度脂蛋白是体内候选的修饰低密度脂蛋白之一，普罗布考具有较强的抗动脉粥样硬化作用。较少

One of the characteristic events in the early lesions of atherosclerosis is the accumulation of lipid-laden foam cells. Recent studies have demonstrated that most of the foam cells are derived from monocute-macrophages. These macrophages accumulate lipids in their cytoplasms, most of which are derived from low density lipoproteins (LDL). Because these cells express very few number of classical LDL receptor, the mechanism of LDL incorporation into macrophages has been a focus of extensive investigation.Goldstein et al. reported that LDL modified chemically by acetylation can be recognized and incorporated by a receptor on macrophages and they named it acetyl-LDL receptor. However, it seems unlikely that such acetylation of LDL would occur in vivo. Therefore, several investigators have been studying what are modified forms of LDL in vivo. Endothelial cell modified LDL, oxidized LDL and cigarette smoke modified LDL are now proposed as a candidate for naturally occurring modified LDL. And … More recently there are lots of evidences that endothelial cell modified LDL is as same as oxidized LDL. Parthasarathy have observed in vitro that LDL converted by chemical oxidation with Cu^<++> or Fe^<++> into a negatively-charged form transforms macrophages into foam cells and suggested that the uptake mechanism of this oxidized LDL by macrophages is mediated by the acetyl-LDL receptor. However in our previous study, we found that acetyl-LDL inhibited the degradation of [^<125>I]oxidized LDL by macrophages only by 55 %. This result has led us to postulate that the uptake mechanisms of the two lipoproteins may be different. 0ur current study was undertaken to examine the above hypothesis by comparing the binding properties of oxidized LDL, cigarette-smoke modified LDL and acetyl-LDL to macrophages. To this, we have performed competition and cross-competition in binding and degradation in macrophages. The results clearly showed that the macrophage receptors for modified LDL are composed of at least three distinct receptors with different specificities. Two of them are specific for those three LDL and the rest is a common receptor for both oxidized and cigarette smoke modified LDL, and acetyl-LDL. In the process of modifying LDL by Cu^<++>, Fe^<++> or cigarette extract, we found that superoxide anion (O_2) played an important role for modification and super-oxide dismutase (SOD) inhibited those reactions. However catalase could not prevent the modification of LDL. Therefore, in stead of S.O.D., we screened out several agents as an antioxidant, such as probucol, vitamin E or vitamin C derivatives.Probucol, so far, is an only agents which could prevent the modification of LDL. Other agents could not prevent those reaction because those could not be included into LDL particles. We next performed in vivo experiment to ask antiatherogenic activity of probucol as an antioxidant using WHHL-rabbits. When rabbits were fed 1 % probucol diet for 17 months since they were 2 month of age, the surface area of atheromatous lesion in aorta was 23 % in comparison with that of 93 % in control WHHL-rabbit. Even when rabbits were fed 1 % probucol diet at 8 month of age when their surface area of lesion was 54 %, 6 month later their lesion became about 38 % on an average. Those date indicated that oxidized LDL is one of the candidate modified LDL in vivo and probucol has a strong antiatherogenic effect. Less