长链非编码RNA-MRAK08352介导神经纤毛蛋白-1在调控静脉桥失效进程中的作用及其机理研究

Although the underlying mechanisms are still incompletely elucidated, thrombosis, neointimal hyperplasia and superimposed atherosclerosis are well recognized as the prime culprits in the development of vein graft failure in the setting of coronary artery bypass grafting. Previously, our study demonstrated a negatively causal relationship between the expression level of long noncoding RNA (LncRNA)-MRAK08352 and neuropilin-1 (Nrp-1) in the progression of a rat vein graft remodeling. Recent studies suggest that Nrp-1, when binding with vascular endothelial growth factor (VEGF) and its receptors, promotes tumor angiogenesis, cellular proliferation and metastases. Although the biological changes inherent to the tumorigenesis and the vein graft remodeling are different, similar cellular and molecular elements may be involved in the vein graft stenotic disease. With regard to our previous findings, we speculate LncRNA-MRAK08352/Nrp-1 signaling might actively participate in the pathogenesis of vein graft failure at different stages. Therefore, in the present study: 1) Structural cells from layers of the vein (eg. smooth muscle cells, fibroblasts, and endothelial cells), vein segments, and rat vein graft models (normal and hyperlipidemia) were used to investigate the functional role and its related signaling pathways of LncRNA MRAK08352/Nrp-1 in the development of vein graft stenotic disease; 2) By using rat vein graft models, a novel VEGF-modulated nanoparticle targeting at LncRNA-MRAK08352 and Nrp-1 is used to evaluate the feasibility and efficacy of this therapeutic strategy. This study may open new horizons in therapeutic interventions aiming at improving long-term vein graft patency in patients undergoing coronary surgery.

血栓形成、新生内膜增生和粥样硬化形成是导致冠脉术后静脉桥失效的主要原因，但其整体调控机制不清。前期研究显示长链非编码RNA (LncRNA)-MRAK08352可调控神经纤毛蛋白-1(Nrp-1)在大鼠静脉管壁重构进程中的表达。Nrp-1可结合VEGF促进肿瘤血管新生和癌细胞增殖扩散，后者与静脉管壁内各层细胞生物学反应行为相似。因此，我们推测Nrp-1可在相关LncRNA调控下积极参与介导静脉桥管壁不同阶段的增殖重构。本研究拟通过分子-细胞-血管环-整体层面，以MRAK08352/Nrp-1为切入点开展研究：1)利用功能获得或缺失实验，确定MRAK08352/Nrp-1在调控静脉桥失效进程中的相关通路机制；2) 在动物模型中，应用VEGF修饰的纳米基因载体调控MRAK08352/Nrp-1在静脉桥管壁重构中各阶段的功能表达，观察该技术的可行性和疗效，为有效防治静脉桥再狭窄治疗开辟新领域。

静脉桥管壁增殖性重构是冠状动脉旁路移植（CABG）术后患者静脉桥失效的根本原因，但具体机制仍需深入研究。我们推测神经纤毛蛋白-1（NRP1）可能在血管内皮细胞(VECs)损伤后新生内膜形成、血管平滑肌细胞(VSMCs)增殖等环节起关键性调控作用；同时，长链非编码RNA（lncRNA）-MRAK083052可通过NRP1介导的多种信号通路调控静脉桥失效。为此本项目开展了以下研究：1）通过靶向干扰NRP1表达，发现NRP1表达的降低可明显抑制大鼠VECs和VSMCs的增殖、迁移并促进细胞凋亡；Western Blot结果提示NRP1通过介导下游的PI3K/Akt、MAPK/ERK及NF-kB信号通路参与调控VECs的生物学功能，介导下游的PI3K/Akt及NF-kB信号通路参与调控VSMCs的生物学功能；进一步利用大鼠单侧颈外静脉-颈总动脉桥接模型进行动物实验，使用N,O-羧甲基化壳聚糖（NOCC）-多醛基透明质酸（A-HA）水凝胶负载NRP1干扰腺病毒敷置于静脉桥表面进行基因转染靶向抑制NRP1表达，发现静脉桥管壁内Ki67的阳性表达指数、静脉桥管壁厚度、内膜/中膜厚度比均显著降低。2）构建过表达MRAK083052病毒载体，通过细胞功能实验发现MRAK083052可明显下调NRP1的表达，同时抑制大鼠VECs和VSMCs的增殖和迁移，并促进其凋亡。通过构建大鼠静脉桥模型，利用NOCC/A-HA水凝胶负载过表达MRAK083052的病毒载体，发现NRP1的表达受到明显抑制，同时静脉桥管壁的增殖性改变也显著降低。3）采用高碘酸钠氧化法合成A-HA，与NOCC交联形成的水凝胶具有明显的三维网状互穿结构和良好的生物可降解性，且对组织无毒副作用和免疫原性； NOCC/A-HA水凝胶可持久稳定地转染静脉桥，其自身的外支架作用及作为基因治疗的有效载体可有效地调控静脉桥失效的整个病理发展过程。综上所述，本研究证明了NRP1在静脉桥失效进程中发挥了重要的促进作用，以NRP1为靶点的治疗策略将有助于防治CABG术后静脉桥再狭窄；NRP1上游的LncRNA-MRAK083052可通过介导NRP1调控静脉桥增殖重构进程，有望成为多层面抑制静脉桥再狭窄不同阶段病理生理变化的新靶点。同时, NOCC/A-HA水凝胶作为新型的基因药物载体，为多层面多阶段动态抑制静脉桥管壁病理性增殖提供了新方法。