新型抗ANGPTL3纳米单抗与SIRPα双功能蛋白通过抑制GSDMD介导的细胞焦亡治疗动脉粥样硬化的作用和机制

动脉粥样硬化（AS）是由异常的脂代谢、炎症和免疫抑制等因素导致的血管内病变，目前尚无十分有效的治疗方法。前期结果表明，抑制血管生成素样蛋白3（ANGPTL3）有望通过控制脂代谢和炎症缓解AS的病程。然而，由于其难以改变斑块中巨噬细胞的免疫抑制状态，因此无法清除斑块。基于此，我们推测：如能在降脂抗炎的同时逆转巨噬细胞的免疫抑制，则有望彻底治疗AS斑块。本课题中，我们首先探究了ANGPTL3在AS中的病理机制并制备了抗ANGPTL3纳米单抗，研究其治疗AS的作用。其次，通过首次将ANGPTL3单抗与CD47的配体SIRPα融合表达制备双功能蛋白，探讨其在降脂抗炎的同时，进一步激活巨噬细胞清除斑块从而治疗AS的作用。最后，通过研究ANGPTL3、巨噬细胞功能与细胞焦亡的关系，以及双功能蛋白治疗后斑块内GSDMD及其相关分子的水平，探讨双功能蛋白通过抑制GSDMD介导的细胞焦亡治疗AS的分子机制。

Atherosclerosis (AS), characterized by progressive lipidic accumulation, inflammation and macrophagic immunosuppression, will ultimately cause the acute cardiovascular events. Although mounting evidence showed that inhibition of angiopoietin-like protein 3 (ANGPTL3) could remarkably attenuate the progression of AS, it still failed to achieve the clearance of the atherosclerotic plaques due to the limited effect of anti-ANGPTL3 therapy on CD47-mediated macrophagic immunosuppression in the atherosclerotic plaque microenvironment. Therefore, we propose that co-targeting ANGPTL3 and macrophagic immunosuppression might be a potential approach for AS therapy. In this project, firstly we plan to investigate the role of ANGPTL3 in AS development, followed by the design of an anti-ANGPTL3 nanobody for AS therapy. Next, the extracellular domain of signal regulatory protein alpha (SIRPα) will be fused to the anti-ANGPTL3 nanobody for endowing the nanobody with the CD47-blocking activity. The therapeutic effect of the novel bifunctional molecule, anti-ANGPTL3 and SIRPα-Fc fusion protein, on AS will then be investigated in ApoE-/- or LDLR-/- atherosclerotic mice, with its role in regulation of lipid metabolism, inflammation and macrophagic immunosuppression to be studied. Moreover, the relationship between ANGPTL3, macrophagic immunosuppression and pyroptosis will be studied in atherosclerotic plaques. The role of Gasdermin D (GSDMD), a key molecule of the pyroptosis, and its-related pathways will be evaluated in ApoE-/- or LDLR-/- mice after they are treated with anti-ANGPTL3 and SIRPα-Fc fusion protein. Our project suggests a novel approach for AS therapy through inhibiting the GSDMD-mediated pyroptosis in atherosclerotic plaques by regulation of lipid metabolism, inflammation and macrophage activity.