Netrin-1 engineered endothelial cell exosomes induce the formation of pre-regenerative niche to accelerate peripheral nerve repair [miRNA-seq]

The formation of vascular niche is pivotal during the early stage of peripheral nerve regeneration. This antecedent angiogenesis meets the nutrient requirements for subsequent rapid axon regeneration and remyelination. Nevertheless, the mechanisms of vascular niche in the regulation of peripheral nerve remain unclear. The axon guidance molecule Netrin-1 (NTN1) is widely expressed in peripheral nerves and particularly was found up-regulated in sciatic nerve stump after peripheral nerve injury (PNI). Herein, we demonstrated that NTN1-high endothelial cells (NTN1+ECs) were the critical component of vascular niche, fostering angiogenesis, axon regeneration and repair-related phenotypes. And we also found that NTN1+ECs derived exosomes (NTN1 EC-EXO) were involved in the formation of vascular niche as a critical role. Multi-omics analysis further verified that NTN1 EC-EXO carried a low-level expression of let7a-5p and activated key pathways associated with niche formation including focal adhesion, axon guidance, PI3K-AKT signaling pathway and mTOR signaling pathway. Taken together, our findings suggested the potential construction of a pre-regenerative niche induced by NTN1 EC-EXO, thereby establishing a conductive microenvironment for nerve repair and facilitating the functional recovery after PNI in the early injury phase.血管微环境（vascular niche）的形成在周围神经再生的早期阶段至关重要。该先期血管生成可满足后续快速轴突再生与髓鞘再生的营养需求。然而，血管微环境在周围神经再生调控中的具体机制仍不明确。轴突导向分子Netrin-1（NTN1）在周围神经中广泛表达，且在周围神经损伤（peripheral nerve injury, PNI）后的坐骨神经残端中表达上调。本研究证实，高表达Netrin-1的内皮细胞（endothelial cells, ECs，NTN1+ECs）是血管微环境的关键组成部分，可促进血管生成、轴突再生及修复相关表型的形成。本研究同时发现，NTN1+ECs来源的外泌体（exosomes, NTN1 EC-EXO）作为关键效应分子参与了血管微环境的构建过程。多组学分析进一步证实，NTN1 EC-EXO携带低表达水平的let7a-5p，并可激活与微环境形成相关的关键信号通路，包括黏着斑（focal adhesion）通路、轴突导向（axon guidance）通路、磷脂酰肌醇3-激酶-蛋白激酶B（PI3K-AKT）信号通路以及哺乳动物雷帕霉素靶蛋白（mTOR）信号通路。综上，本研究结果表明，NTN1 EC-EXO可诱导构建预再生微环境，从而为神经修复建立有利微环境，并促进周围神经损伤后早期阶段的功能恢复。