EphrinB2反向信号在动静脉内瘘术后静脉血管壁成熟中的作用机制研究

Arteriovenous fistula (AVF) is the main surgical therapy in maintenance hemodialysis for the patients with the end-stage renal disease. However, excessive venous intimal hyperplasia and insufficient venous outward remodeling happen frequently after the operation, which leads to high morbidity of venous stenosis and occlusion and corresponding poor prognosis. EphB4, a receptor tyrosine kinase, and its ligand, EphrinB2, are important in angiogenesis and various functions of vascular cells; the receptor EphB4 can bind with ligand EphrinB2 and activate EphrinB2 reverse signaling. Our previous research indicated that EphrinB2/EphB4 are important in venous maturation after AVF, EphB4 expression inhibition leads excessive venous intimal hyperplasia after AVF and the expression of EphrinB2, a marker of arterial endothelial cells, increases in venous endothelial cells after AVF; our pre-experiment found that EphrinB2 reverse signaling is related to Akt1and eNOS signaling in immortalized mouse lung endothelial cells and human umbilical vein endothelial cell, and has a marked impact on venous outward remodeling and intimal hyperplasia after AVF. In this project, to clarify the function and mechanism of EphrinB2 reverse signaling to venous maturation after AVF and to Akt1-eNOS-NO signaling pathway in vascular endothelial cells, clinical research, in vitro experiment with arterial and venous endothelial cell and in vivo experiment with EphrinB2iEC-KO mouse aorta-inferior vene cava fistula model are designed, lentivirus infection technique to induce or inhibit the expression of EphrinB2 and some specific methods to stimulate or inhibit EphrinB2 reverse signaling are used, Akt1 inhibitors and eNOS inhibitors are used to inhibit the related downstream signals. Furthermore, clarification of the function and regulation pathway of EphrinB2 reverse signaling may provide a new theoretical evidence for drug prevention of venous stenosis and occlusion and to increase the maturation rate of AVF.

动静脉内瘘手术（AVF）后静脉成熟率低下是血液透析治疗亟待解决的难题之一。受体EphB4激活配体EphrinB2的反向信号开始受到人们的重视。我们前期研究发现，EphrinB2/EphB4信号通路在AVF术后静脉成熟中发挥重要作用，EphrinB2反向信号对AVF术后静脉血管扩张和内膜增生影响显著，并与血管内皮细胞（vEC）中Akt1、eNOS信号通路相关，但机制尚不清楚。本项目以临床患者、vEC和血管内皮细胞EphrinB2敲除小鼠为研究对象；通过临床标本分析，vEC过表达或抑制表达EphrinB2，刺激、抑制、干扰EphrinB2反向信号，抑制下游Akt1、eNOS活性以及研究小鼠腹主动脉-下腔静脉AVF模型等方法，旨在阐明EphrinB2反向信号通过vEC内Akt1-eNOS-NO信号通路调控vEC功能，影响AVF术后静脉成熟的作用和机制，为提高AVF术后静脉成熟率探索新的治疗靶点。

本课题项目瞄准动静脉内瘘手术（AVF）后静脉成熟率低下这一血液透析治疗亟待解决的难题。分别通过体内实验和体外实验，在小鼠腹主动脉-下腔静脉AVF模型与鼠、人来源的血管内皮细胞中使用Ephb4-Fc激活EphrinB2反向信号，并分别检测Akt1-eNOS-NO 信号通路的激活情况，初步证实EphrinB2反向信号的激活，通过活化Akt1-eNOS-NO 信号通路，促进小鼠AVF术后静脉血管的适应性重塑（血管直径扩张及血管壁的增厚），同时调控内皮细胞的增殖、迁移、内皮管的形成与NO的释放等，有望成为AVF术后成熟过程中新的干预靶点。此外，对人体来源的AVF术后样本与人正常头静脉的生物信息学分析和实验验证，鉴定了一批在AVF术后成熟中发挥潜在作用的免疫相关基因。由于客观原因及不可抗力，课题方向调整后，研究人员瞅准同领域（同肾病-透析领域）的另一大难题——同种/异种异体血管移植在动静脉透析通路中应用和机制研究，成功构建了首个大鼠颈部脱细胞血管移植物同种异体AVG血管移植模型与首个大鼠颈部脱细胞血管移植物异种（羊肋间动脉）异体AVG血管移植模型，并对模型进行了组织学和免疫学上的评估。有望解决脱细胞血管移植物在AVG应用领域长期缺乏合适动物模型，机制方面研究进展缓慢的痛点，同时，在此动物模型上开展的机制研究正在进行中，模型的成功建立为后续的研究奠定了有力基础。