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）：单核细胞衍生的巨噬细胞与人类和实验模型中的动脉粥样硬化斑块的起始和进展至关重要。牙菌斑破裂被认为是人类急性冠状动脉综合征的触发事件，已经描述了几只小鼠的斑块破裂模型。巨噬细胞通过释放稳定的蛋白酶与牙菌斑破裂有关。巨噬细胞凋亡在晚期动脉粥样硬化病变中很突出，但是巨噬细胞凋亡是有益还是有害是有争议的。在此提案中，我们将检查实验性动脉粥样硬化中巨噬细胞凋亡的机制和后果。我们建议巨噬细胞在内膜中的积累和激活有助于平滑肌细胞死亡，从而促进斑块破裂。因此，我们将测试主要假设，即巨噬细胞凋亡降低了内膜病变的进展，并最终有助于斑块稳定。作为次要假设，我们还提出，Fasedied信号传导在动脉粥样硬化中的巨噬细胞凋亡和激活中起重要作用。在AIM 1中，我们将使用同位素编码的亲和力TAG和与质谱法的串联亲和力纯化来确定体外调节激活与凋亡的fas信号传导复合物的组成部分。在AIM 2中，我们将确定凋亡蛋白在体外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，BCL-2，显性阴性FADD和p35 caspase抑制剂，对FAS结扎或动脉粥样硬化刺激的巨噬细胞激活或凋亡的影响。在AIM 3中，我们将确定巨噬细胞凋亡对LDLR - / - 和APOE-/ - 小鼠病变进展和斑块破裂的封锁作用。我们将用新型的逆看病毒载体转导造血干细胞（HSC），该载体构成了巨噬细胞限制的人CD68启动子，并编码1个抗凋亡蛋白。然后，我们将确定用编码1个抗凋亡蛋白的1个病毒转导的HSC的移植对早期和晚期病变和LDLR - / - 小鼠中的斑块破裂的影响。 LDLR - / - 模型中转导HSC移植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，通过在巨噬细胞中选择性地过表达BCL-2。