A study on oxidative modification of low-density lipoprotein and the response of vascular endothelial cells

低密度脂蛋白氧化修饰及血管内皮细胞反应的研究

• 批准号: 14370792
• 负责人: OKADA Masahiko
• 金额: $ 7.1万
• 依托单位: Niigata University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14370792/
• 关键词: oxidized LDL; VCAM-1; mRNA; endothelial cell; IL-1; prediction of 3D structure; α-helix; lipid monolayor; mRNA; IL-1; 立体構造予測; αヘリックス; 脂質一重膜; 非特異的たんぱく質; トリプシン; エピトープ解析; 動脈硬化症

Radical species cause oxidative modification of low-density lipoprotein(LDL), which plays a key role in the very early stage of atherogenesis. However, the exact mechanism how oxidized LDL acts as an initiator of atherosclerosis and what causes abonormal signals in the endothelial cells are not still clear. In this study, we conducted experiments as how LDL is oxidized, what kind of modification plays an important role for the endothelial cells.First we examined the structure of sugar chain on the LDL particles and identified eight types of sugar chains. There was no significant difference between native LDL and oxidized LDL in the structure. When we released mannose by adding an enzyme, a significant prolongation of the lag phase was observed Second we analyzed the amino acid sequence of each fragment that was generated by oxidation of LDL, and found that various serum proteins such as apolipoprotein A-I, fibrin, α2-macrqglobulin, haptoglobin, etc. were non-specifically bound to LDL. … More Finally we established a method to purify thus contaminated LDL by using gel permeation chromatography, and obtained purified fragments of oxidized LDL.We already found that vascular endothelial adhesion molecule-1(VCAM-1) expressed on the endothelial cells by the stimulation of oxidized LDL. VCAM-1 plays a key role to adhere macophages in the every early stage of atherogenesis. So we conducted experiments to examine whether the level of messenger RNA(mRNA) of VCAM-1 would rise or not by adding purified oxidized LDL. First we added IL-1 to the endothelial cells by changing its concentration and incubation time. It was found that two hours incubation was the most effective time to induce mRNA of VCAM-1. Finally we succeeded in finding the most appropriate condition and observing the induction of mRNA of VCAM-1 by adding fragments of oxidized LDL.Another result of the study was to find a method to estimate three-dimensional structure of apolipoprotein B-100(apoB-100). Because of its extraordinary size and insoluble nature, the three dimensional structure has been difficult to deduce. The method was developed based on the consideration that sites of α-helices with higher hydrophilicity are more likely to combine with each other and form bundles in the non-polar environment. Two cylinder models were established and the interactive force (E) between them was calculated according to hydrophobicity and the charge on the amino acids. Consequently, a series of values of E ranging from 383 to 7315 was obtained. Extremely high E values were considered to represent regions of α-helices. If we combined the regions in close proximity to each other, 5 candidates for α-helix bundles were identified. These regions matched well with the lipid-binding motifs of apoB-100 as reported by others. Thus, our algorithm was useful in predicting the higher-order structure of apoB-100, and proper use of this method may enable identification of potential lipid-associating domains of membrane proteins. Less

自由基会引起低密度脂蛋白（LDL）的氧化修饰，这在动脉粥样硬化形成的早期阶段发挥着关键作用。然而，氧化低密度脂蛋白作为动脉粥样硬化引发剂的确切机制以及导致内皮细胞信号异常的原因尚不清楚。在这项研究中，我们进行了实验来了解LDL是如何被氧化的，什么样的修饰对内皮细胞起重要作用。首先，我们检查了LDL颗粒上的糖链结构，并鉴定了八种类型的糖链。天然LDL和氧化LDL在结构上没有显着差异。当我们通过添加酶释放甘露糖时，观察到滞后期显着延长。其次，我们分析了LDL氧化产生的每个片段的氨基酸序列，发现各种血清蛋白如载脂蛋白A-I、纤维蛋白、α2-巨球蛋白、触珠蛋白等与LDL非特异性结合。 … 更多 最后，我们建立了一种利用凝胶渗透色谱纯化被污染的LDL的方法，并获得了纯化的氧化LDL片段。我们已经发现，在氧化LDL的刺激下，血管内皮粘附分子-1（VCAM-1）在内皮细胞上表达。 VCAM-1 在动脉粥样硬化形成的每个早期阶段都在粘附巨噬细胞方面发挥着关键作用。因此，我们进行了实验来检测通过添加纯化的氧化LDL是否会升高VCAM-1的信使RNA(mRNA)水平。首先，我们通过改变其浓度和孵育时间将IL-1添加到内皮细胞中。结果发现，孵育2小时是诱导VCAM-1 mRNA最有效的时间。最后我们成功地找到了最合适的条件，并观察了通过添加氧化LDL片段对VCAM-1 mRNA的诱导。研究的另一个结果是找到了一种估计载脂蛋白B-100（apoB-100）三维结构的方法。由于其非凡的尺寸和不可溶的性质，三维结构一直难以推断。该方法的开发基于考虑到亲水性较高的α-螺旋位点在非极性环境中更容易相互结合并形成束。建立了两个圆柱体模型，根据疏水性和氨基酸的电荷计算它们之间的相互作用力（E）。因此，获得了从383到7315的一系列E值。极高的 E 值被认为代表 α 螺旋区域。如果我们将彼此非常接近的区域组合起来，就会识别出 5 个 α 螺旋束的候选区域。这些区域与其他人报道的 apoB-100 的脂质结合基序非常匹配。因此，我们的算法可用于预测 apoB-100 的高阶结构，并且正确使用该方法可以识别膜蛋白的潜在脂质相关结构域。较少的

项目成果

A new method of measuring the antioxidant activity of polyphenols using cumene hydroperoxidase.

使用枯烯氢过氧化物酶测量多酚抗氧化活性的新方法。

• 发表时间: 2004
• 期刊: Ann.Clin.Biochem. 41
• 作者: O.Sugita;N.Ishizawa;T.Matsuto;M.Okada;N.Kayahara
• 通讯作者: N.Kayahara

T13M mutation of lecithin-cholesterol acyltranseferase gene causes fish-eye disease.

卵磷脂胆固醇酰基转移酶基因T13M突变导致鱼眼病。

• 发表时间: 2004
• 期刊: Clin.Chim.Acta 343
• 作者: T.Miida;B.Zhang;K.Obayashi;Y.Seino;Y.Zhu;T.Ito;Y.Nakamura;M.Okada;K.Saku
• 通讯作者: K.Saku

Risk of carotid artery atherosclerosis : a prospective study in non-diabetic subjects (Niigata Study)

颈动脉粥样硬化的风险：非糖尿病受试者的前瞻性研究（新泻研究）

• 发表时间: 2003
• 期刊: AHA Second Asia Pacific Scientific Forum (Final Program)
• 作者: Takahata K;Katsuki H;Kume T;Nakata D;Ito K;Muraoka S;Yoneda F;Kashii S;Honda Y;Akaike A.;T.Miida;M.Okada
• 通讯作者: M.Okada

Seitaro Maruyama: "Fenofibrate, a peroxisome proliferator-activated receptor α activator, suppresses experimental autoimmune myocarditis by stimulating the interleukin-10"J. Atheroscler. Thromb.. 9・2. 87-92 (2002)

Seitaro Maruyama：“非诺贝特，一种过氧化物酶体增殖物激活受体 α 激活剂，通过刺激白细胞介素 10 抑制实验性自身免疫性心肌炎”J. Thromb.. 87-92。

Macroscopic assessment of skin venules in patients with familial amyloidotic polyneuropathy(FAP) using near-infrared spectrophotoscopy, as a marker of autonomic dysfunction.

使用近红外分光光度法对家族性淀粉样变性多发性神经病（FAP）患者的皮肤小静脉进行宏观评估，作为自主神经功能障碍的标志。

• 发表时间: 2004
• 期刊: Amyloid and Amyloidosis.(G.Gilles ed.)(CRC Press)
• 作者: K.Obayashi;Y.Ando;T.Miida;M.Nakamura;T.Yamashita;M.Ueda;K.Haraoka;H.Terazaki;M.Okada
• 通讯作者: M.Okada