血管壁微环境Oxysterol/Ox-LDL通过氧化固醇结合蛋白ORP4L诱导巨噬细胞凋亡的机制研究

Atherosclerosis is a chronic inflammatory disease, in which macrophages play a central role. Apoptosis of macrophage foam cells is suggested to trigger plaque destabilization at the final phase of the disease. Oxysterols/Ox-LDL are major components of the pathologic microenvironments in the blood vessel wall which induce macrophage apoptosis, but the molecular mechanisms underlying macrophage apoptosis remain incompletely understood. As revealed by our previous study, ORP4L exists in CD68 positive macrophages in arterial lesions and interacts physically with phospholipase C-β3 (PLCβ3). ORP4L knock-down regulates PLCβ3 activity in control of IP3/Ca2+ signaling and cell survival. Through changing the conformation of ORP4L, 25-hydroxycholesterol has in macrophages similar effects on PLCβ3 function and IP3 production as ORP4L knock-down. Consistently, PLCβ3 deficiency has been shown to lead to macrophage hypersensitivity to apoptosis and reduction of atherosclerosis in mice (J Clin Invest. 2008 Jan;118(1):195-). It was previously found that a constitutive low level of ITPR-mediated Ca2+ release is essential for optimal cell bioenergetics and viability (Cell 142(2):270-83). We hypothesize that ORP4L, as a cytosolic oxysterol receptor, could play a central role in macrophage apoptosis via controlling PLCβ3 activity and IP3/Ca2+ signaling in the blood vessel wall. This project will focus on the role of ORP4L in mediating the induction of macrophage apoptosis by oxysterols/Ox-LDL in the pathologic microenvironments of blood vessel wall. ORP4L-/- mice and molecular/cell biology and bioinformatic approaches are employed to address the detailed mechanisms of macrophage apoptosis.

氧化固醇/氧化低密度脂蛋白是血管壁病理微环境主要组分，参与诱导巨噬细胞凋亡，但机制不清。我们发现，氧化固醇结合蛋白ORP4L存在于小鼠腹腔和动脉斑块巨噬细胞，在THP-1巨噬细胞中与PLCβ3物理共存，并调控PLCβ3活性、IP3产生、钙离子节律和细胞凋亡。氧化固醇改变ORP4L的构像，显示了类似ORP4L沉默对巨噬细胞PLCβ3活性和IP3产生的抑制作用。PLCβ3被在体实验证实能改变巨噬细胞凋亡敏感性并影响动脉粥样硬化发展；内质网释放的钙离子节律被证实是维持线粒体呼吸链正常功能和ATP产生的必要条件。据此推测，氧化固醇/氧化低密度脂蛋白通过结合其细胞内受体ORP4L，抑制PLCβ3/IP3/Ca2+信号途径，导致血管壁巨噬细胞发生线粒体途径凋亡。本课题拟利用我们产生的ORP4L-/-小鼠，多层次探讨ORP4L介导氧化固醇/氧化低密度脂蛋白对巨噬细胞凋亡的作用，明确其在血管壁的凋亡机制。

巨噬细胞凋亡是动脉粥样硬化形成和发展的中心事件。氧化低密度脂蛋白及其中负载的氧化固醇诱导细胞凋亡是否有相关的受体蛋白分子参与的问题仍有待揭示。我们研究首次发现，作为细胞内氧化固醇的受体，ORP4L建立的钙离子信号通路对维持巨噬细胞的生存至关重要。血管微环境中过量蓄积的氧化固醇/氧化低密度脂蛋白通过结合ORP4L，抑制ORP4L的正常功能，诱导巨噬细胞凋亡。我们的研究提供了一个氧化固醇/氧化低密度脂蛋白细胞毒性的机制模型。通过基因敲除小鼠模型，证明ORP4L是影响动脉粥样硬化发生发展的关键蛋白。我们从分子、细胞、组织和整体动物水平多层次验证了该理论，为动脉粥样硬化过程中巨噬细胞的凋亡提供机制解释，加深人们对血管损伤及动脉粥样硬化发生、发展的理解。ORP4L可能成为干预动脉粥样硬化疾病进程的重要靶标。