Multimerin 2通过拮抗C1q/RAGE通路抑制动脉粥样硬化发生研究

The receptor of advanced glycation end-product (RAGE) and subsequent pathway plays an important role in the pathogenesis of atherosclerosis. Using co-immunoprecipitation approach and proteomics analysis and with verification, we found that complement C1q and multimerin 2 proteins were competitive in binding with RAGE. The serum C1q levels were significantly higher, but multimerin 2 levels were significantly lower in patients with coronary artery disease than in controls, with both levels being associated with disease severity. Peritoneal injection of C1q recombinant protein promoted atherogenesis in apoE-deficient mice. Multimerin 2, in contrast, inhibited C1q-induced pro-inflammatory effects in endothelial cells, and endothelium-macrophage interaction. These results suggest that multimerin 2 exerts atherosclerosis-inhibitory effect through potential aspects including antagonization of C1q-RAGE interaction. In future study, we will investigate the biological effects of multimerin 2 regarding its antagonizing influence on C1q-RAGE interaction, using microarray analysis, co-immunoprecipitation, yeast two-hybrid technique, radioligand receptor binding assay, and adenovirus transfection in cell and animal experiments. The site of C1q-RAGE-multimerin2 interaction will be ascertained.

终末糖化产物受体（RAGE）通路与动脉粥样硬化（AS）发生关系密切。我们用蛋白组学实验发现并证实，C1q和multimerin 2均与RAGE结合，两配体间存在竞争。冠心病患者血清C1q升高而multimerin 2降低，两者水平与冠脉病变严重性相关。C1q促进小鼠AS发生；而multimerin 2抑制C1q诱导的内皮细胞炎症反应及内皮细胞-单核巨噬细胞交互作用。结合文献提示multimerin 2可能通过包括拮抗C1q-RAGE结合及其促炎效应等环节来抑制AS发生。后续将利用免疫共沉淀、酵母双杂交、放射性配体受体结合、芯片分析、腺病毒过表达等技术从细胞和动物整体水平验证multimerin 2抑制AS效应，明确multimerin 2拮抗C1q/RAGE通路和干预AS发生机制，揭示C1q-RAGE-multimerin2交互作用、结合的效能和位点。从而阐明multimerin 2抑制AS的作用及机制。

动脉粥样硬化是心脑血管事件发生的病理生理基础。内皮屏障功能受损和血管内膜通透性增加是较早的表现，将有助于单核巨噬细胞跨内皮转移，加剧动脉炎症反应的发生。Multimerin-2 (MMRN2)是一种主要表达于血管表面的细胞外基质蛋白，但是否与血管内皮屏障功能及通透性有关、与动脉粥样硬化的发生有无关联还不清楚。课题组主要从组织和动物整体层面研讨了MMRN2与血管内皮屏障功能以及动脉粥样硬化的关系。以血管内皮细胞和小鼠为研究对象，从体外细胞学水平研究外源性重组MMRN2蛋白对炎症因子导致的人脐静脉内皮细胞（HUVEC）的通透性改变的作用，以及炎症因子和血管通透性相关分子表达的作用；从整体小鼠水平研究MMRN2对炎症因子导致的主要脏器血管通透性改变的影响；研究MMRN2对ApoE敲除小鼠动脉粥样硬化的作用。实验结果发现：LPS和TNF-α分别使HUVEC单层通透性增加，而MMRN2抑制这一效应。Western blot检测显示LPS和TNF-α使炎症因子（IL-6、COX-2和TNF-α）表达上调，而MMRN2剂量依赖性降低这些炎症因子的表达。Western blot显示LPS和TNF-α使P-MLC、ROCK2、P-MYPT表达增加，而MMRN2剂量依赖性降低这些内皮通透性相关因子的表达，表明MMRN2通过影响内皮细胞MLC磷酸化从而调节血管内皮通透性。LPS腹腔注射分别使C57BL/6小鼠的内脏血管通透性增加，MMRN2可以抑制小鼠脏器血管通透性增加，然而MMRN2基因敲除可以使小鼠内脏血管通透性增加更为显著。免疫荧光检测和western blot检测发现，有粥样硬化病变的冠状动脉较人正常内乳动脉MMRN2表达量显著减少。重组MMRN2蛋白腹腔注射使ApoE基因敲除小鼠的主动脉动脉粥样硬化斑块显著减轻，同时小鼠主动脉血管通透性减少；然而，MMRN2和ApoE基因双敲除小鼠的主动脉动脉粥样硬化斑块显著加重，同时主动脉血管通透性增加更为严重。通过本研究发现，MMRN2可以抑制血管内皮通透性增加，并且抑制动脉粥样硬化的发生。本课题的完成将有助于阐明MMRN2改善动脉粥样硬化的重要作用和机制，将可能为动脉粥样硬化的防治提供新的靶点和治疗策略。