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巨噬细胞免受oxldl诱导的线粒体功能障碍和细胞死亡，从而降低动脉粥样硬化的严重程度。巨噬细胞和泡沫细胞死亡在动脉粥样硬化病变的发展中起着至关重要的作用。我们建议研究谷胱甘肽还原酶介导的巨噬细胞肿瘤保护的分子机制。特异性目的1：确定OxLDL对线粒体硫醇氧化还原状态的影响。我们的初步数据表明，OxLDL诱导线粒体去极化和ATP合成的损失。我们将使用人类单核细胞来源的巨噬细胞来确定OxLDL是否通过以下途径促进肿瘤发生：1)改变线粒体的硫醇氧化还原状态；2)使线粒体谷胱甘肽还原酶失活；3)增加线粒体内膜通透性。特异性目的2：确定线粒体和细胞质谷胱甘肽还原酶在预防氧化低密度脂蛋白诱导的肿瘤中的作用。线粒体不合成谷胱甘肽（GSH），因此依靠谷胱甘肽的摄取和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细胞质谷胱甘肽还原酶（GR）的表达是否能阻止GSSG积累和蛋白硫醇氧化；2)增加谷胱甘肽还原酶活性是否能恢复线粒体功能并保护巨噬细胞免受氧化低密度脂蛋白诱导的肿瘤。特异性目的3：确定增加巨噬细胞谷胱甘肽还原酶活性是否降低动脉粥样硬化的严重程度。泡沫细胞死亡促进坏死核心的形成和动脉粥样硬化病变的进展。我们将进行骨髓移植研究，以确定巨噬细胞中谷胱甘肽还原酶（GR）的增强表达是否能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过表达gr的骨髓细胞将用于重新填充受辐照的LDL受体缺失小鼠和apoE缺失小鼠。我们将测量病变大小和病变胆固醇/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.