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（由申请方提供）：单核细胞衍生的巨噬细胞在人类以及实验模型中与动脉粥样硬化斑块的发生和进展密切相关。斑块破裂被认为是人类急性冠状动脉综合征的触发事件，并且已经描述了几种斑块破裂的小鼠模型。巨噬细胞通过释放去稳定化蛋白酶参与斑块破裂。巨噬细胞凋亡在晚期动脉粥样硬化病变中很突出，但巨噬细胞凋亡是有益的还是有害的还存在争议。在这个建议中，我们将研究实验性动脉粥样硬化形成中巨噬细胞凋亡的机制和后果。我们认为巨噬细胞在内膜中的积聚和活化导致平滑肌细胞死亡，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主要假设。作为次要假设，我们还提出Fas介导的信号转导在动脉粥样硬化形成中巨噬细胞凋亡和活化中起重要作用。在目标1中，我们将确定Fas信号复合物的组件调节激活与凋亡的巨噬细胞在体外，使用同位素编码的亲和标签和串联亲和纯化与质谱。在目标2中，我们将确定凋亡蛋白在体外巨噬细胞活化和凋亡中的作用。我们将研究抗凋亡蛋白c-FLIP、Bcl-2、显性负性FADD和p35半胱天冬酶抑制剂对Fas连接或致动脉粥样硬化刺激引起的巨噬细胞活化或凋亡的影响。在目标3中，我们将确定阻断巨噬细胞凋亡对LDLR-/-和ApoE-/-小鼠中病变进展和斑块破裂的影响。我们将用一种新的逆转录病毒载体转染造血干细胞（HSC），该载体整合了巨噬细胞限制性人CD 68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之一的病毒转导的HSC移植对LDLR-/-小鼠中早期和晚期病变和斑块破裂的影响。通过在巨噬细胞中产生选择性过表达Bcl-2的转基因ApoE-/-小鼠，将在LDLR-/-模型中移植转导HSC的阳性结果扩展至ApoE-/-模型中的病变起始/进展和斑块破裂。