岩藻糖基化对PlexinD1调控泡沫细胞迁出斑块的作用及机制

Atherosclerosis (AS) is a lipid settled in artery intima, macrophage foaming and other comprehensive chronic inflammatory disease. The dynamic changes of macrophage migration plays a key role in AS occurrence, progression and prognosis, but the regulatory mechanism is not fully understand. It is one of the prerequisite on the AS regression that foamy macrophage migration is restored, and swum out of AS plaque. Our research work shows that neuroimmune guidance cue receptor PlexinD1 fucosylation may affect the activation by its ligands Sema3E, then regulating foamy macrophage to swim out the AS plaque. This project will use methods, like fucosidase hydrolysis, lectin blot and so on, to evaluate if fucosyltransferase could catalyze receptor PlexinD1 fucosylation to regulate on macrophage migration, in Apoe-/- mouse model of AS plaque progression and regression, and a foam cell model in vitro. Using technologies of glycobiology and single point mutation to determine which glycosylation site of PlexinD1 is closely related to its function. And we will demonstrate the corresponding molecular mechanisms through FRET and downstream signaling pathways analysis in this project. It will reveal the novel regulation ways on AS regression. It is of benefit to understand AS clinical outcome and provide a highlight therapy target.

动脉粥样硬化（AS）是脂质沉积动脉内膜、巨噬细胞泡沫化等综合性慢性炎症疾病。巨噬细胞迁移力的动态变化对AS发生、发展及转归起着关键作用，但调控机制不完全清楚。恢复斑块内泡沫细胞的迁移力，诱导泡沫细胞游出是AS斑块消退的重要前提之一。课题组前期工作显示，神经导向因子受体PlexinD1岩藻糖基化可能影响配基Sema3E对其的激活，从而调控泡沫细胞游出斑块。本项目将利用Apoe-/-小鼠斑块进展模型、斑块消退以及体外泡沫细胞模型，用岩藻糖苷酶水解、lectin blot等技术证明岩藻糖基转移酶能否通过修饰PlexinD1来调控巨噬细胞迁移；用糖生物学、点突变等技术确定与其功能密切相关的糖基化位点；运用FRET等技术及下游信号通路分析来研究岩藻糖基化调控PlexinD1功能的机制。以期揭示AS斑块消退调控新的重要通路，为临床上认识AS转归与合理治疗提供新思路。

动脉粥样硬化是一种以大量脂质和泡沫细胞堆积在内膜下为特征的慢性炎症性疾病。泡沫细胞滞留斑块是AS损害的标志性事件，修复巨噬-泡沫细胞的运动能力是促进斑块消退的重要手段。神经导向因子Sema3E及其受体PlexinD1被认为是促进泡沫细胞滞留于斑块的因子。因受体多为糖蛋白，蛋白糖基化修饰对于蛋白的折叠、构型、稳定性及功能具有重要作用。PlexinD1作为膜受体蛋白，糖基化修饰对其蛋白功能的调控以及对细胞迁移能力的作用，目前尚未见报道。本项目证实了巨噬细胞泡沫化后，其迁移能力显著下降，且PlexinD1及其配体Sema3E表达上调。PlexinD1在Fut8催化下可发生α-1, 6核心岩藻糖基化修饰。在本项目中，我们证实了泡沫化巨噬细胞的迁移能力受PlexinD1的表达及其α-1, 6核心岩藻糖基化水平调控。当PlexinD1过表达、其与配体的结合被抗体封闭后或者Fut8被敲低后，均可使泡沫化巨噬细胞迁移能力恢复。我们进一步利用ApoE-/-动脉粥样硬化模型小鼠，证实了PlexinD1、Fut8的表达水平与动脉粥样斑块形成、进展及消退紧密相关，其原因是可能因为PlexinD1和Fut8参与调控巨噬源性泡沫细胞迁移能力，影响泡沫细胞在斑块内的沉积。而其下游信号通路可能与CDC42-PAK相关。本项目从受体糖基化的角度，揭示泡沫细胞游出内膜促进斑块消退的可能途径。初步阐明与泡沫细胞运动相关的受体 PlexinD1 糖基化修饰对其移出斑块的重要作用。为进一步探索动脉粥样硬化的炎症诱发机制和血管炎症损伤疾病的防治提供一个新的治疗靶点及思路。