Lipid macropinocytosis: a novel target in atherosclerotic cardiovascular disease

项目摘要 动脉粥样硬化是全世界死亡的主要潜在原因，其特征在于慢性炎症， 脂质在动脉壁中的积聚。巨噬细胞（MΦ）和血管脂质过度蓄积 平滑肌细胞（SMC）在动脉粥样硬化的发生和发展中起关键作用。一般 接受动脉粥样硬化是由于动脉壁中LDL的修饰及其随后的内在化引起的 通过多种清道夫受体被MΦ和SMCs激活。清道夫受体非依赖性脂质的作用 动脉粥样硬化中的摄取，刺激这一过程的生理因素和信号机制 所涉及的问题仍然没有得到很好的描述。我们报道了基质蛋白血小板反应蛋白-1（TSP 1）刺激 在MΦ中直接、不依赖于清道夫受体摄取未修饰的天然LDL（nLDL）。TSP 1通过其同源物 受体CD 47激活肌动蛋白结合蛋白cofilin，导致nLDL的巨胞饮作用， 胆固醇积累。刺激巨胞饮作用的CD 47下游的信号传导机制是 未知尽管磷脂酶C（PLC）和弹弓磷酸酶1（SSH 1）已被证明激活 cofilin，它们在TSP 1诱导的巨胞饮中的作用尚未研究。此外， TSP 1-CD 47信号刺激动脉粥样硬化血管MΦ巨胞饮及其意义 脂质巨胞饮在动脉粥样硬化发病机制中的作用仍有待确定。我们的小说初稿 数据显示，TSP 1和CD 47敲除小鼠免受动脉粥样硬化和巨胞饮作用的影响， 抑制剂EIPA降低高胆固醇血症小鼠动脉粥样硬化病变形成。我们假设 TSP 1通过CD 47促进动脉壁中的脂质巨胞饮作用，从而促进脂质积聚， 动脉粥样硬化的发病机制。本研究将通过以下几个方面来检验这一假设：（1）检验第一个假设 MΦs中CD 47受体信号转导是否通过PLC和SSH 1介导的cofilin刺激巨胞饮作用的时间 激活并有助于动脉粥样硬化的发展;（2）探索MΦ是否通过 体内动脉粥样硬化动脉巨胞饮和EIPA主要靶点NHE 1特异性缺失 在MΦs中减弱动脉粥样硬化，以及（3）研究TSP 1与CD 47结合是否刺激 通过Nox 1介导的cofilin激活在MΦ样SMC中的巨胞饮作用。该提案将利用全球和 细胞特异性基因敲除小鼠和其他遗传工具来验证这一假设。通过多种途径特异性靶向CD 47 方法（抗体阻断、siRNA/吗啉代沉默和CD 47活化肽序列）将 确认在遗传突变体中获得的结果。将使用多种互补技术， 在体外（药理学、遗传学、荧光/高分辨率显微镜）和体内研究巨胞饮作用 （cofilin突变体，AngioSPARK 680，MΦ特异性NHE 1敲除）。这一创新性建议有可能 揭示了脂质内化的重要新机制，并为我们对脂质内化的认识提供了范式转变。 动脉粥样硬化是如何发展的

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.