转录因子KLF4介导的新型多肽H-KI20抑制视网膜新生血管的分子机制

Retinal neovascularization is the leading cause of blindness in a lot of ocular fundus diseases. Numerous therapeutic strategies targeting vascular endothelial growth factor (VEGF) are being studied. However, the large molecular weight and extremely high annual treatment drug cost have limited their wide use. Previously, we screened out a novel peptide with small molecular weight, H-KI20, which is able to effectively inhibit retinal neovascularization in mice. It was easy to prepare with low cost, and also had strong activity and stability (acquired national patent recently). Through Gene Chip assay, we further found that H-KI20 down regulate the expression of transcription factor KLF4 which was intimately related to neovascularization. In this study, we plan to investigate the role of KLF4 in the antiangiogenic molecular mechanisms of H-KI20，to observe the activity of H-KI20 in the KLF4 knock down or over expression retinal vascular endothelial cell model; To detect the change of transcription factor binding sites and gene regulatory network with ChIP-seq technique , especially the change of binding sites with Vegf gene promoter, and how it contributes to the H-KI20 anti-neovascularization activity mediated by KLF4. This work will lay the foundation for clinical translation of this small size peptide H-KI20 to safe, efficient, economic, and novel antiangiogenic inhibitor in retinal neovascular treatment.

视网膜新生血管是诸多致盲性眼底病的关键病理变化，治疗棘手。已有的抗血管内皮生长因子（VEGF）药物分子量大、成本高。课题组从肝细胞生长因子中筛选并优化得到新型小分子肽H-KI20，能有效抑制小鼠视网膜新生血管生成，合成简便、成本低（近期获国家发明专利）；通过基因芯片筛选并验证H-KI20显著下调与血管新生密切相关的转录因子KLF4表达。本项目拟进一步研究KLF4在H-KI20抗新生血管机制中的作用；观察H-KI20在KLF4基因敲低或过表达的视网膜血管内皮细胞模型中抗血管生成活性的变化，应用染色质免疫共沉淀-测序（ChIP-seq）技术检测H-KI20干预下KLF4的转录因子结合谱变化，尤其是与Vegf基因启动子结合位点的变化，探索其基因调控通路，深入揭示KLF4介导的H-KI20抗新生血管分子机制；并为H-KI20转化为机制明确、安全有效、成本经济的新型视网膜新生血管抑制剂奠定理论基础。

课题组前期自主研发的抗眼部新生血管小分子肽H-KI20能有效抑制视网膜血管的新生。依托本课题我们进一步针对H-KI20的药物代谢动力学和抑制血管生成的分子机制做了深入研究，创新性地提出JNK/ATF2信号转导通路是H-KI20抑制VEGF刺激的视网膜血管新生的重要靶点。研究内容包括H-KI20的体外稳定性、穿细胞膜能力、穿透血眼屏障的能力、抑制内皮细胞迁移能力、H-KI20滴眼后动物视网膜组织中的代谢动力学，以及H-KI20抑制VEGF下游与血管生成相关的信号转导通路。经实验发现：H-KI20在多种缓冲液中具有较高的稳定性；除了具有良好的细胞膜穿透能力， H-KI20对小鼠模型滴眼0.5 h后，RPE -脉络膜-巩膜复合体和视网膜中的平均浓度分别达到33.8 ng/mL和20.5 ng/mL，证明H-KI20具有较佳的血眼屏障穿透能力；在对H-KI20抑制血管生成的分子机制研究中发现：H-KI20对VEGF刺激导致的JNK磷酸化有显著抑制作用；VEGF刺激HRECs中磷酸化的ATF2表达，加入H-KI20后，ATF2的活化被抑制；运用计算机辅助分子对接模拟技术评估了29个JNK和/或其他上游调控ATF2的蛋白与H-KI20的对接得分，发现H-KI20与JNK2的相互作用评分最高，模拟对接提示JNK2中的Gln28、Ala53、Arg69、Glu73、Ile148、Asp151、Asp169残基分别通过氢键与H-KI20中的Lys20、Thr15、Ile1、Ile14、Lys9、Ser7、Ser13结合，且JNK2中Asp169羧基的氧原子与H-KI20中Arg6胍基的氮原子形成盐桥。表面等离子共振技术（SPR）证明H-KI20与JNK2分子之间具有良好的结合力。研究结果表明H-KI20通过下调JNK/ATF2信号通路中JNK2和ATF2的表达和活化发挥抗新生血管的重要作用。本研究成果有利于深入明确抗新生血管多肽H-KI20的分子调控网络机制，为推进H-KI20作为一种安全稳定的小分子肽在糖尿病视网膜病变等新生血管性眼病治疗中的应用做出良好铺垫。依托本项目，已发表SCI期刊论著4篇，1篇论著已被眼科权威SCI期刊Invest Ophthalmol Vis Sci接收待刊，培养毕业博士研究生两名，即将毕业博士研究生一名。

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