Department of Cardiology, Pan-Vascular Research Institute of Tongji University, Shanghai Tenth People's Hospital, Tongji University School of Medicine, Shanghai, China.
Aab Cardiovascular Research Institute and Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, USA.
Br J Pharmacol. 2022 Apr;179(8):1716-1731. doi: 10.1111/bph.15743. Epub 2022 Jan 11.
The Dll4-Notch1 signalling pathway plays an important role in sprouting angiogenesis, vascular remodelling and arterial or venous specificity. Genetic or pharmacological inhibition of Dll4-Notch1 signalling leads to excessive sprouting angiogenesis. However, transcriptional inhibitors of Dll4-Notch1 signalling have not been described.
We designed a new peptide targeting Notch signalling, referred to as TAT-ANK, and assessed its effects on angiogenesis. In vitro, tube formation and fibrin gel bead assay were carried out, using human umbilical vein endothelial cells (HUVECs). In vivo, Matrigel plug angiogenesis assay, a developmental retinal model and tumour models in mice were used. The mechanisms underlying TAT-ANK activity were investigated by immunochemistry, western blotting, immunoprecipitation, RT-qPCR and luciferase reporter assays.
The amino acid residues 179-191 in the G-protein-coupled receptor-kinase-interacting protein-1 (GIT1-ankyrin domain) are crucial for GIT1 binding to the Notch transcription repressor, RBP-J. We designed the peptide TAT-ANK, based on residues 179-191 in GIT1. TAT-ANK significantly inhibited Dll4 expression and Notch 1 activation in HUVECs by competing with activated Notch1 to bind to RBP-J. The analyses of biological functions showed that TAT-ANK promoted angiogenesis in vitro and in vivo by inhibiting Dll4-Notch1 signalling.
We synthesized and investigated the biological actions of TAT-ANK peptide, a new inhibitor of Notch signalling. This peptide will be of significant interest to research on Dll4-Notch1 signalling and to clinicians carrying out clinical trials using Notch signalling inhibitors. Furthermore, our findings will have important conceptual and therapeutic implications for angiogenesis-related diseases.
Dll4-Notch1 信号通路在发芽血管生成、血管重塑以及动脉或静脉特异性中发挥重要作用。Dll4-Notch1 信号的遗传或药理学抑制会导致过度发芽血管生成。然而，尚未描述 Dll4-Notch1 信号的转录抑制剂。
我们设计了一种针对 Notch 信号的新型肽，称为 TAT-ANK，并评估了其对血管生成的影响。在体外，使用人脐静脉内皮细胞（HUVEC）进行管形成和纤维蛋白凝胶珠测定。在体内，使用 Matrigel plugs 血管生成测定、发育性视网膜模型和小鼠肿瘤模型。通过免疫化学、Western blot、免疫沉淀、RT-qPCR 和荧光素酶报告基因测定研究了 TAT-ANK 活性的机制。
G 蛋白偶联受体-激酶相互作用蛋白-1（GIT1-ankyrin 结构域）中的氨基酸残基 179-191 对于 GIT1 与 Notch 转录抑制因子 RBP-J 的结合至关重要。我们基于 GIT1 中的残基 179-191 设计了肽 TAT-ANK。TAT-ANK 通过与激活的 Notch1 竞争与 RBP-J 结合，显著抑制 HUVEC 中 Dll4 的表达和 Notch1 的激活。生物学功能分析表明，TAT-ANK 通过抑制 Dll4-Notch1 信号促进体外和体内血管生成。
我们合成并研究了 TAT-ANK 肽的生物学作用，这是一种新的 Notch 信号抑制剂。该肽将对 Dll4-Notch1 信号研究以及使用 Notch 信号抑制剂进行临床试验的临床医生具有重要意义。此外，我们的发现将对与血管生成相关的疾病具有重要的概念和治疗意义。
