Macrophage Apoptosis in Atherogenesis

动脉粥样硬化形成中的巨噬细胞凋亡

• 批准号: 7039213
• 负责人: JOHN Marshall HARLAN
• 金额: $ 37.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7039213
• 关键词: BCL2 gene /protein; apolipoprotein E; apoptosis; atherosclerotic plaque; genetically modified animals; hematopoietic stem cells; laboratory mouse; leukocyte activation /transformation; low density lipoprotein receptor; macrophage; mass spectrometry; pathologic process; proteolysis; stem cell transplantation; transfection; vascular smooth muscle

DESCRIPTION (provided by applicant): Monocyte-derived macrophages are critically involved in the initiation and progression of the atherosclerotic plaque in man as well as in experimental models. Plaque rupture is thought to be the trigger event for acute coronary syndromes in man, and several mouse, models of plaque rupture have been described. Macrophages have been implicated in plaque rupture by releasing de-stabilizing proteases. Macrophage apoptosis is prominent in advanced atherosclerotic lesions, but it is controversial whether macrophage apoptosis is beneficial or detrimental. In this proposal we will examine the mechanisms and consequences of macrophage apopotosis in experimental atherogenesis. We propose that macrophage accumulation and activation in the intima contributes to smooth muscle cell death and thereby promotes plaque rupture. Therefore, we will test the primary hypothesis that macrophage apoptosis reduces intimal lesion progression and ultimately contributes to plaque stabilization. As secondary hypotheses, we also propose that Fasmediated signaling plays an important role in macrophage apoptosis and activation in atherogenesis. In Aim 1, we will identify components of the Fas signaling complex regulating activation versus apoptosis in macrophages in vitro, using isotope-coded affinity tag and tandem affinity purification with mass spectrometry. In Aim 2, we will determine the role of apoptosis proteins in macrophage activation and apoptosis in vitro. We will investigate the effect of the anti-apoptotic proteins, c-FLIP, Bcl-2, dominant-negative FADD, and p35 caspase inhibitor, on macrophage activation or apoptosis in response to Fas ligation or atherogenic stimuli. In Aim 3, we will determine the effect of blockade of macrophage apoptosis on lesion progression and plaque rupture in LDLR-/-and ApoE-/-mice. We will transduce hematopoietic stem cells (HSCs) with a novel retrbviral vector incorporating the macrophage-restricted human CD68 promoter and encoding 1 of the antiapoptotic proteins. We will then determine the effect of transplantation of HSCs transduced with a virus encoding 1 of these anti-apoptotic proteins on early and advanced lesions and plaque rupture in LDLR-/- mice. Positive results with transplantion of transduced HSC in the LDLR-/-model will be extended to lesion initiation/progression and plaque rupture in the ApoE-/-model by generating transgenic ApoE-/- mice overexpressing Bcl-2 selectively in macrophages.

描述(由申请人提供)：单核细胞来源的巨噬细胞在人类动脉粥样硬化斑块的启动和发展中起关键作用，在实验模型中也是如此。斑块破裂被认为是人类急性冠脉综合征的触发事件，已经描述了几种斑块破裂的小鼠模型。巨噬细胞通过释放不稳定的蛋白水解酶参与斑块破裂。巨噬细胞凋亡在晚期动脉粥样硬化病变中是显著的，但巨噬细胞凋亡是有益的还是有害的还存在争议。在这项提案中，我们将研究实验性动脉粥样硬化中巨噬细胞凋亡的机制和后果。我们认为，巨噬细胞在血管内膜中的聚集和激活有助于平滑肌细胞的死亡，从而促进斑块破裂。因此，我们将检验巨噬细胞凋亡减少内膜病变进展并最终有助于斑块稳定的基本假设。作为第二个假说，我们还提出Fas介导的信号在巨噬细胞的凋亡和激活中在动脉粥样硬化形成中起重要作用。在目标1中，我们将使用同位素编码的亲和标签和串联亲和纯化与质谱仪相结合的方法，在体外鉴定调节巨噬细胞激活和凋亡的Fas信号复合体的成分。在目标2中，我们将在体外确定凋亡蛋白在巨噬细胞激活和凋亡中的作用。我们将研究抗凋亡蛋白c-flip、bc-2、显性阴性FADD和p35半胱氨酸天冬氨酸氨基转移酶抑制剂在Fas结扎或动脉粥样硬化刺激下对巨噬细胞激活或凋亡的影响。在目标3中，我们将确定阻断巨噬细胞凋亡对LDLR-/-和ApoE-/-小鼠病变进展和斑块破裂的影响。我们将用一种新型逆转录病毒载体转导造血干细胞(HSCs)，该载体含有巨噬细胞限制性人CD68启动子，并编码1种抗凋亡蛋白。然后，我们将确定用编码这些抗凋亡蛋白中的一种的病毒转导的HSC移植对LDLR-/-小鼠早期和晚期病变和斑块破裂的影响。在LDLR-/-模型中移植转导的HSC的阳性结果将扩展到ApoE-/-模型中的病变起始/进展和斑块破裂，方法是产生选择性地在巨噬细胞中过表达Bcl2的转基因ApoE-/-小鼠。