Role of Glutathione Reductase and Macrophage Oncosis

描述(由申请人提供)：我们假设增加谷胱甘肽还原酶活性可以保护人类巨噬细胞免受OxLDL诱导的线粒体功能障碍和细胞死亡的影响，从而降低动脉粥样硬化的严重程度。巨噬细胞和泡沫细胞的死亡在动脉粥样硬化病变的发展中起着至关重要的作用。我们拟研究谷胱甘肽还原酶介导的巨噬细胞抗肿瘤作用的分子机制。具体目的1：确定氧化低密度脂蛋白对线粒体硫醇氧化还原状态的影响。我们的初步数据表明，氧化低密度脂蛋白诱导线粒体去极化和ATP合成的丧失。我们将使用人类单核细胞来源的巨噬细胞来确定OxLDL是否通过1)改变线粒体的硫醇氧化还原状态，2)失活线粒体谷胱甘肽还原酶和3)增加线粒体内膜通透性来促进肿胀。线粒体不合成谷胱甘肽(GSH)，因此依赖于GSH的摄取和GSSG的还原来维持适当的硫醇氧化还原状态。我们将使用人类单核细胞来源的巨噬细胞来确定1)腺病毒介导的多西环素控制的线粒体或胞浆谷胱甘肽还原酶(GR)的表达是否可以防止GSSG积聚和蛋白质硫醇氧化；2)增加谷胱甘肽还原酶活性是否可以恢复线粒体功能并保护巨噬细胞免受OxLDL诱导的肿瘤的影响。具体目标3：确定巨噬细胞谷胱甘肽还原酶活性增加是否降低动脉粥样硬化的严重程度。泡沫细胞死亡促进了坏死核的形成和动脉粥样硬化病变的进展。我们将在体内进行骨髓移植研究，以确定巨噬细胞中谷胱甘肽还原酶(GR)的增强表达是否可以防止泡沫细胞死亡和病变进展。通过逆转录病毒基因转移产生的高表达GR的骨髓细胞将被用于重新繁殖受辐射的低密度脂蛋白受体缺失小鼠和载脂蛋白E缺失小鼠。我们将测量病变大小和病变的胆固醇/胆固醇酯含量。

DESCRIPTION (provided by applicant): We hypothesize that increased glutathione reductase activity protects human macrophages from OxLDL-induced mitochondrial dysfunction and cell death, thereby decreasing the severity of atherosclerosis. Macrophage and foam cell death by oncosis plays a crucial role in the development of atherosclerotic lesions. We propose to study the molecular mechanism of glutathione reductase-mediated protection of macrophages from oncosis.Specific Aim 1: To determine the effect of OxLDL on the thiol redox state of mitochondria. Our preliminary data demonstrate that OxLDL induces mitochondrial depolarization and loss of ATP synthesis. We will use human monocyte-derived macrophages to determine if OxLDL promotes oncosis by 1) altering the thiol redox status of mitochondria, 2) inactivating mitochondrial glutathione reductase and 3) increasing mitochondrial inner membrane permeability.Specific Aim 2: To determine the role of mitochondrial and cytosolic glutathione reductase in preventing OxLDL-induced oncosis. Mitochondria do not synthesize glutathione (GSH) and therefore rely on GSH uptake and the reduction of GSSG to maintain the appropriate thiol redox state. We will use human monocyte-derived macrophages to determine 1) if adenovirus-mediated doxycycline-controlled expression of mitochondrial or cytosolic glutathione reductase (GR) prevents GSSG accumulation and protein thiol oxidation and 2) if increasing glutathione reductase activity restores mitochondrial function and protects macrophages from OxLDL-induced oncosis.Specific Aim 3: To determine whether increased macrophage glutathione reductase activity decreases the severity of atherosclerosis. Foam cell death promotes the formation of the necrotic core and the progression of atherosclerotic lesions. We will perform bone marrow transplantation studies to determine in vivo whether augmented expression of glutathione reductase (GR) in macrophages prevents foam cell death and lesion progression. GR-overexpressing bone marrow cells, generated by retroviral gene transfer, will be used to repopulate irradiated LDL receptor null mice and apoE null mice. We will measure both lesion size and lesional cholesterol/cholesterol ester content.