LRG1/GDF-15促血管新生在动脉粥样硬化斑块进展中的作用及机制

Aberrant neovascularization is a key feature of vulnerable plaque formation contributing to plaque rupture. In our previous study, we showed that growth differentiation factor-15 (GDF-15) was closely associated with atherosclerosis and acute myocardial infarction (AMI) (JACC 2017). The results suggested that LRG1/GDF-15 signaling is key regulator in the process of angiogenesis in atherosclerosis. However, the mechanisms underlying excessive angiogenesis associated with atherosclerosis is not fully understood. In this study, therefore, ApoE knock-out and ApoE/LRG1 double knock-out mouse model have been used, and were treated with LRG1 inhibition or supplementation in vivo and in vitro. To investigate the morphology of plaque, calculate vulnerability index and observe the migration, proliferation and tube formation of vascular endothelial cells. Here we explore the role of the modulating function of LRG1 to angiogenic signaling involves GDF-15/ALK1/Smad and VEGF/Ang/Tie2, and inflammatory factors involves NF-κB and TNF-α in aberrant pathological angiogenesis in atherosclerosis. Our objectives in this study, therefore, were to illustrate the new mechanism of LRG1/GDF-15 promoting angiogenesis in atherosclerotic plaque, the atherogenic process and the formation of vulnerable plaque. It has a prospective future and far-reaching significance on the target to provide theoretic and experimental basis for identifying high risk coronary artery disease patients early.

血管新生为动脉粥样硬化易损斑块形成、破裂的主要诱发因素。前期研究我们发现生长分化因子-15(GDF-15)与动脉粥样硬化和急性心肌梗死发病密切相关(JACC 2017)但其机制尚未清楚。我们推测富亮氨酸α2糖蛋白1(LRG1)/GDF-15信号通过调控血管新生在动脉粥样硬化进展起重要作用。在此基础上，我们拟利用LRG1基因敲除(KO)和ApoE/LRG1 DKO动脉粥样硬化动物模型，通过LRG1体内外干预实验，观测斑块病理形态和易损指数改变，分析血管内皮细胞增殖、迁移和体外成管变化，系统探讨LRG1因子对GDF-15/ALK1/Smad及VEGF/Ang/Tie2血管新生信号及NF-κB等炎症信号的调控作用，阐明LRG1/GDF-15信号促动脉粥样硬化血管新生、动脉粥样硬化进展及易损斑块形成中的新机制，为冠心病高危患者提供早期预警指标。

炎症反应为动脉粥样硬化易损斑块形成、破裂的主要诱发因素。本项目我们主要探讨了LRG1/GDF15信号通路对动脉粥样硬化炎症反应及易损斑块形成的调控作用。通过分析人颈动脉斑块LRG1表达量、分布情况和血浆标本LRG1含量，发现在冠心病患者人群中LRG1明显升高，同时在动脉粥样硬化组织中表达明显增加，通过免疫组化观察，LRG1在动脉粥样硬化斑块血管中较正常血管明显增加，进一步探讨LRG1基因缺失对动脉粥样硬化易损斑块及相关炎症信号的影响，在 ApoeKO和Apoe/Lrg1 DKO小鼠模型上高脂喂养诱导动脉粥样硬化斑块形成，测定了血压水平、血脂水平、促炎症因子/抗炎症因子、氧化应激、GDF15、NO水平的检测，完成了流式粒细胞分选实验。探讨了LRG1基因缺失情况下对炎症因子、氧化应激以及动脉粥样硬化改变情况，并取得了较好的研究成果。研究发现与ApoeKO组小鼠相比，ApoE/LRG1 DKO小鼠动脉粥样硬化病变明显增加，炎症反应增强，血管促炎症因子(TNF-α、TNFRSF1A、IL-1β、IL-6、MCP-1等)的生成、NOX4、TGF-β1与eNOS磷酸化水平均明显上调，伴有斑块增多，脂质核心增大，和细胞焦亡现象。说明了LRG1对动脉粥样硬化进展的影响这一科学问题。按照研究计划通过构建Lenti-Cas9-LRG1-sgRNA质粒，开展巨噬细胞刺激实验、单核-内皮细胞粘附试验，应用LRG1及LPS、IL-4等诱导剂刺激干预巨噬细胞，发现LRG1可以促进巨噬细胞向M1型巨噬细胞表型分化，进一步制备了LRG1-中和抗体，发现LRG1中和抗体E4C12，可以显著抑制LRG1诱导的巨噬细胞极化。进而将E4C12应用于小鼠体内，观察动脉粥样硬化进展程度，发现E4C12可以显著抑制动脉粥样硬化进展。因此本研究阐明 LRG1 调控炎症反应延缓动脉粥样硬化进展的分子机制。LRG1通过激活巨噬细胞，促进巨噬细胞向促炎M1型巨噬细胞分化，加速动脉粥样硬化的进展，LRG1中和抗体可以显著抑制巨噬细胞分化，抑制动脉粥样硬化进展，因此LRG1有望成为动脉粥样硬化治疗新机制和新靶点。