Lipid macropinocytosis: a novel target in atherosclerotic cardiovascular disease

脂质巨胞饮作用：动脉粥样硬化性心血管疾病的新靶点

• 批准号: 10172967
• 负责人: Gabor Csanyi
• 金额: $ 38.5万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10172967
• 关键词: Actin-Binding Protein; Affinity; Antibodies; Antigen Presentation; Aorta; Apolipoprotein E; Area; Arterial Fatty Streak; Arteries; Atherosclerosis; Attenuated; Binding; Blocking Antibodies; CD36 gene; CD47 gene; Cardiovascular system; Cause of Death; Cell Fractionation; Cell membrane; Cells; Cholesterol; Chronic; Confocal Microscopy; Data; Development; Dissociation; Exhibits; Extracellular Matrix Proteins; Flow Cytometry; Fluorescence; Foam Cells; Genetic; High Pressure Liquid Chromatography; Human; In Vitro; Inflammation; Inflammatory; Knock-out; Knockout Mice; Knowledge; Link; Lipid-Laden Macrophage; Lipids; Lipoproteins; Low Density Lipoprotein Receptor; Low Density Lipoprotein oxidation; Low-Density Lipoproteins; Mediating; Membrane; Microscopy; Modification; Mus; NHE1; Other Genetics; Pathogenesis; Pathway interactions; Pharmacology; Phenotype; Phospholipase C; Phosphoric Monoester Hydrolases; Physiological; Play; Process; Protein Dephosphorylation; Protein Isoforms; Proteins; Receptor Signaling; Reporting; Resolution; Role; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Subendothelial Layer; Techniques; Testing; Thrombospondin 1; Time; United States; Vascular Smooth Muscle; Western Blotting; Woman; cofilin; cost; health care delivery; in vivo; inhibitor/antagonist; innovation; macrophage; men; mouse genetics; mutant; nanoparticle; novel; overexpression; platelet-derived growth factor BB; protein aminoacid sequence; receptor; scavenger receptor; therapeutic target; tool; uptake; vascular inflammation

Project Summary Atherosclerosis, the main underlying cause of death worldwide, is characterized by chronic inflammation and accumulation of lipids in the arterial wall. Excessive lipid accumulation by macrophages (MΦs) and vascular smooth muscle cells (SMCs) plays a key role in the initiation and progression of atherosclerosis. It is generally accepted that atherosclerosis arise from LDL modification in the arterial wall and its subsequent internalization by MΦs and SMCs through a variety of scavenger receptors. The role of scavenger receptor-independent lipid uptake in atherosclerosis, the physiological factors stimulating this process, and the signaling mechanisms involved remain poorly characterized. We reported that matrix protein thrombospondin-1 (TSP1) stimulates direct, scavenger receptor-independent uptake of unmodified, native LDL (nLDL) in MΦs. TSP1 via its cognate receptor CD47 activates actin-binding protein cofilin, leading to macropinocytosis of nLDL, and excessive cholesterol accumulation. The signaling mechanisms downstream of CD47 that stimulate macropinocytosis are unknown. Although phospholipase C (PLC) and slingshot phosphatase 1 (SSH1) have been shown to activate cofilin, their roles in TSP1-induced macropinocytosis have not yet been investigated. Moreover, the ability of TSP1-CD47 signaling to stimulate MΦ macropinocytosis in atherosclerotic vessels in vivo and the significance of lipid macropinocytosis in the pathogenesis of atherosclerosis remain to be determined. Our novel preliminary data show that TSP1 and CD47 knockout mice are protected from atherosclerosis and the macropinocytosis inhibitor EIPA decreases atherosclerotic lesion formation in hypercholesterolemic mice. We hypothesize that TSP1 via CD47 promotes lipid macropinocytosis in the arterial wall, contributing to lipid accumulation and the pathogenesis of atherosclerosis. The hypothesis will be tested via the following aims: (1) examining for the first time whether CD47 receptor signaling in MΦs stimulates macropinocytosis via PLC- and SSH1-mediated cofilin activation and contributes to atherosclerosis development; (2) exploring whether MΦs internalize lipoproteins via macropinocytosis in atherosclerotic arteries in vivo and that deletion of NHE1 (major target of EIPA) specifically in MΦs attenuates atherosclerosis, and (3) investigating whether TSP1 binding to CD47 stimulates macropinocytosis in MΦ-like SMCs via Nox1-mediated cofilin activation. The proposal will employ global and cell-specific knockout mice, and other genetic tools to test the hypothesis. Specific targeting of CD47 via multiple approaches (antibody blockade, siRNA/morpholino silencing, and CD47 activating peptide sequences) will provide confirmation of results obtained in genetic mutants. Multiple complementary techniques will be used to study macropinocytosis in vitro (pharmacological, genetic, fluorescence/high-resolution microscopy) and in vivo (cofilin mutants, AngioSPARK 680, MΦ-specific NHE1 knockouts). This innovative proposal has the potential to reveal important new mechanisms of lipid internalization and provide a paradigm shift in our knowledge about how atherosclerosis develops.

项目摘要 动脉粥样硬化是世界范围内死亡的主要潜在原因，其特点是慢性炎症和 动脉壁中脂质的积聚。巨噬细胞过度脂质蓄积(MΦS)与血管 平滑肌细胞(SMC)在动脉粥样硬化的发生发展中起着关键作用。它一般都是 公认动脉粥样硬化是由动脉壁中低密度脂蛋白的修饰及其随后的内化引起的 由MΦ、S和SMC通过多种清道夫受体介导。清道夫受体非依赖性脂质的作用 动脉粥样硬化的摄取、刺激这一过程的生理因素和信号机制 涉案人员的特征仍然很差。我们报道了基质蛋白血栓反应蛋白-1(TSP1)刺激 MΦS TSP1通过其同源物直接非依赖清道夫受体摄取未经修饰的天然低密度脂蛋白 受体CD47激活肌动蛋白结合蛋白cofilin，导致nLDL的巨噬细胞吞噬，并过度 胆固醇积聚。CD47下游刺激巨噬细胞吞噬的信号机制有 未知。尽管磷脂酶C(PLC)和弹弓磷酸酶1(SSH1)已被证明激活 Cofilin，它们在TSP1诱导的巨噬细胞吞噬中的作用尚未被研究。更重要的是， TSP1-CD47信号刺激动脉粥样硬化血管巨噬细胞吞噬M-Φ及其意义 脂质巨噬细胞增多在动脉粥样硬化发病机制中的作用尚不明确。我们的小说初步 数据显示，TSP1和CD47基因敲除小鼠可免受动脉粥样硬化和巨噬细胞增多症的影响 抑制剂EIPA减少高胆固醇血症小鼠动脉粥样硬化病变的形成。我们假设 TSP1通过CD47促进动脉壁中脂质的巨噬细胞吞噬，有助于脂质堆积和 动脉粥样硬化的发病机制。该假说将通过以下目的进行检验：(1)检验第一个 M-Φ中CD47受体信号是否通过PLC和SSH_1介导的聚集刺激巨噬细胞增多的时间 激活并参与动脉粥样硬化的发展；(2)探讨MΦS是否通过 动脉粥样硬化中巨噬细胞吞噬和特异性缺失NHE1(EIPA的主要靶点) 在MΦ中，S减轻动脉粥样硬化；(3)研究TSP 1与CD47结合是否刺激 巨噬细胞吞噬M-Φ样细胞通过NOX1介导的cofilin激活。该提案将雇用全球和 细胞特异性基因敲除小鼠和其他基因工具来检验这一假说。CD47的特异性靶向通过多个 方法(抗体阻断、siRNA/吗啉沉默和CD47激活肽序列)将 提供对在基因突变中获得的结果的确认。将使用多种互补技术来 在体外和体内研究巨噬细胞吞噬作用(药理学、遗传学、荧光/高分辨率显微镜) (cofilin突变体，AngioSPARK680，MΦ特异性NHE1基因敲除)。这项创新的建议有可能 揭示了重要的脂质内化新机制，并提供了我们对 动脉粥样硬化是如何发展的。