Role of Ca^<2+> signaling and reactive oxygen species in endothelial dysfunction induced by lysophosphatidylcholine

Ca^2信号和活性氧在溶血磷脂酰胆碱诱导的内皮功能障碍中的作用

• 批准号: 13670732
• 负责人: ISHIBASHI Toshiyuki
• 金额: $ 2.18万
• 依托单位: Fukushima Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13670732/
• 关键词: Endothelial cells; Calcium; Rho; Reactive oxygen species; nonselective cation current; lipid modification; geranylgeranylation; statins; H_2O_2; 膜電流; パッチクランプ; リゾフォスファチジルコリン; HMG-CoA還元酵素阻害薬

Lysophosphatidylcholine (LPC) is an atherogenic compound of oxidized low density lipoprotein which evokes endothelial dysfunction. In this research project, we investigated the roles of Ca^<2+> and reactive oxygen species (ROS) in signal transduction induced by LPC in endothelial cells (Ecs).LPC is known to increase intracellular Ca^<2+> concentration ([Ca^<2+>]_i) Ecs. [Ca^<2+>]_i was determined in cultured human aortic Ecs by fura-2 assay and membrane current was measured by whole-cell patch-clamp. HMG-CoA reductase inhibitors (statins) markedly decreased the [Ca^<2+>]_i increase caused by LPC. This suppressive effect was quickly reversed by geranylgeranylpyrophosphate (GGPP) and was mimicked by inhibitors of Rho and Rho-kinase. LPC induced the translocation of the GTP-bound active form of RhoA into membranes within 1 minute as determined by pull-down assay and reduced the levels of RhoA in the cytoplasm, indicating that LPC quickly activates RhoA. Statins prevented the GTP/GDP excha … More nge of RhoA and its membrane translocation from the cytoplasm caused by LPC and these effects of statins were reversed by GGPP. The responses of RhoA activation to statins and GGPP concurred with their effects on Ca^<2+> mobilization. LPC also induced a nonselective cation current (NSC) after a lag. Statins prolonged the lag and decreased the current amplitude and GGPP abolished the inhibitory effect of statins on the current. In summary of this part, LPC induced Ca^<2+> mobilization and membrane current via a Rho activation-dependent PLC pathway in Ecs and statins blocked these effects by preventing the GGPP-dependent lipid modification of Rho. The present study implicates Rho in LPC stimulation of Ca^<2+> movement (Yokoyama K, Ishibashi T et al. Circulation. 2002 ; 105 : 962-967). In addition, the effect of LPC on membrane current and its signaling were examined in cardiomyocytes (Li L et al. Mol Pharmacol. 2002 : 62 ; 602-607).We also investigated the association of ROS with Ca^<2+> influx in LPC stimulation. The [Ca^<2+>]_i increase caused by LPC was suppressed by radical scavengers such as superoxide dismutase (SOD) and catalase. In fact, we detected the generation of hydrogen peroxide (H_2O_2) 60 seconds after LPC stimulation. When external Ca^<2+> was present, the suppressive effect of catalase was greater than that of SOD. Catalase, but not SOD, suppressed the amplitude of NSC induced by LPC. H_2O_2 indeed induced NSC in Ecs. We show that in LPC stimulation H_2O_2 contributes to NSC in Ecs, suggesting that H_2O_2 generated by LPC may play a role in Ca^<2+> signaling mediated via NSC.Furthermore, we studied the role of RhoA activation in the gene expression of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1) in human macrophages (Atherosclerosis. 2002 ; 163 : 39-47, BBA. 2002 ; 1590 : 123-130). Less

溶血磷脂酰胆碱（LPC）是氧化低密度脂蛋白的致动脉粥样硬化化合物，可引起内皮功能障碍。在本研究项目中，我们研究了Ca^<2+>和活性氧（ROS）在LPC诱导内皮细胞（Ecs）信号转导中的作用。已知LPC增加细胞内Ca^<2+>浓度（[Ca^<2+>]_i） Ecs。用fura-2法测定人主动脉内皮细胞[Ca^<2+>]_i，用全细胞膜片钳法测定膜电流。HMG-CoA还原酶抑制剂（他汀类）可显著降低LPC引起的[Ca^<2+>]_i升高。这种抑制作用很快被香叶基焦磷酸（GGPP）逆转，并被Rho和Rho激酶抑制剂模拟。通过下拉实验，LPC在1分钟内诱导gtp结合的RhoA活性形式易位到膜中，并降低了细胞质中RhoA的水平，表明LPC快速激活了RhoA。他汀类药物阻止GTP/GDP交换，使LPC引起的RhoA和其膜从细胞质中移位，而他汀类药物的这些作用被GGPP逆转。RhoA活化对他汀类药物和GGPP的反应与它们对Ca^<2+>动员的影响一致。LPC还在一段滞后后诱导非选择性阳离子电流（NSC）。他汀类药物延长了滞后期，降低了电流振幅，GGPP消除了他汀类药物对电流的抑制作用。综上所述，在Ecs中，LPC通过Rho激活依赖的PLC途径诱导Ca^<2+>动员和膜电流，而他汀类药物通过阻止ggpp依赖的Rho脂质修饰来阻断这些作用。目前的研究暗示Rho在LPC刺激Ca^<2+>运动（Yokoyama K， Ishibashi T等）。流通。2002；105: 962-967)此外，我们还研究了LPC对心肌细胞膜电流及其信号传导的影响（Li L等）。医学杂志。2002:62；602 - 607)。我们还研究了在LPC刺激中活性氧与Ca^<2+>内流的关系。LPC引起的[Ca^<2+>]_i升高被超氧化物歧化酶（SOD）和过氧化氢酶等自由基清除剂抑制。事实上，我们在LPC刺激后60秒检测到过氧化氢（H_2O_2）的生成。当外源Ca^<2+>存在时，过氧化氢酶的抑制作用大于SOD。过氧化氢酶抑制LPC诱导的NSC的幅度，而SOD没有。H_2O_2确实诱导了Ecs的NSC。我们发现在LPC刺激下，H_2O_2对Ecs的NSC有促进作用，提示LPC产生的H_2O_2可能在NSC介导的Ca^<2+>信号通路中起作用。此外，我们研究了RhoA激活在人巨噬细胞组织因子（TF）和纤溶酶原激活物抑制剂-1 （PAI-1）基因表达中的作用(动脉粥样硬化；2002;163:39-47，BBA。2002年;1590: 123-130)。少

项目成果

Ishibashi T: "Integral role of RhoA activation in monocyte adhesion-triggered tissue factor expression in endothelial cells"Arteriosclerosis, Thrombosis, and Vascular Biology. 23. 681-687 (2003)

Ishibashi T：“RhoA 激活在内皮细胞中单核细胞粘附触发的组织因子表达中的整体作用”动脉硬化、血栓形成和血管生物学。

吉田雅幸: "動脈硬化と炎症、スタチンの新たなる抗動脈硬化作用"血管医学. 2. 383-390 (2001)

Masayuki Yoshida：“动脉硬化和炎症，他汀类药物的新抗动脉硬化作用”《血管医学》2. 383-390 (2001)。

Li L: "Inhibitory effect of fluvastatin on lysophosphatidylcholine-induced nonselective cation current in Guinea pig ventricular myocytes"Molecular Pharmacology. 62(3). 602-607 (2002)

李丽：“氟伐他汀对豚鼠心室肌​​细胞溶血磷脂酰胆碱诱导的非选择性阳离子电流的抑制作用”分子药理学。

Yokoyama K: "HMG-CoA reductase inhibitors suppress intracellular calcium mobilization and membrane current induced by lysophophatidylcholine in endothelial cells"Circulation. 105. 962-967 (2002)

Yokoyama K：“HMG-CoA还原酶抑制剂抑制内皮细胞中溶血磷脂酰胆碱诱导的细胞内钙动员和膜电流”循环。

Li L: "Inhibitory effect of fluvastatin on lysophosphatidlcholine-induced nonselective cation current in Guinea-pig ventricular myocytes"Molecular Pharmacology. 62. 602-607 (2002)

李丽：“氟伐他汀对豚鼠心室肌​​细胞溶血磷脂胆碱诱导的非选择性阳离子电流的抑制作用”分子药理学。