AGEs诱导单核巨噬细胞外泌体内miRNA改变在糖尿病内皮功能紊乱中的作用及机制研究

Endothelial dysfunction is one of the most important causes of diabetic vascular diseases. Resent studies show that exosomes (EXs) can modulate cell-cell communication and function, which play a pivotal role in diabetic vascular diseases. Our previous data show that advanced glycation end products (AGEs) induce monocyte/macrophage EXs (M-EXs) released, and impair angiogenesis. Moreover, our previous data show that the levels of miR-22-5p upregulated and let-7f-1-3p downregulated in the M-EXs by miRNA array analysis, and both of them are related to angiogenesis and endothelial function. However, in progress of diabetic vascular diseases, the roles of monocyte/ macrophage -derived EXs still unknown, the role of miRNAs and their target gene need further exploration. Based on literature and our previous data, we hypothesized that AGEs can impair angiogenesis and endothelial function in diabetic condition via regulate monocyte/macrophage EXs release and change miR-22-5p and let-7f-1-3p level in M-EXs. Moreover, in this proposed application, we will use different approaches such as gene knockout mice and RNA interference to understand how M-EXs tranduct the miRNAs to endothelial cell and regulate their target gene which play an important role in the accelerated progression of diabetic vascular diseases. The results generated from the proposed study lays a novel foundation for control and prevention of diabetic vascular disease.

内皮功能紊乱是糖尿病血管病变的主要早期危险因素。外泌体（EXs）是近年来研究细胞间信息交流和功能调控的热点。我们前期工作表明：EXs亦参与了糖尿病血管并发症进展；晚期糖基化终产物（AGEs）可促进单核巨噬细胞来源EXs（M-EXs）的释放，且M-EXs可损伤内皮细胞管腔形成能力；miRNA芯片发现M-EXs中多种miRNA水平异常（血管功能相关的miR-22-5p升高、let-7f-1-3p降低）。然而AGEs介导的M-EXs释放及其携带的miRNA改变在糖尿病血管病变中的作用尚不明确。本项目将从细胞水平、基因敲除动物模型及临床研究等层面验证我们的假设：AGEs促进M-EXs释放及其携带内皮功能密切相关的miRs改变，进而介导内皮细胞中miR-22-5p及let-7f-1-3p表达异常，通过调控相关靶基因导致内皮功能紊乱。研究结果有望为糖尿病血管病变防治提供新的靶点。

内皮功能紊乱（endothelial dysfunction）存在于糖尿病血管病变早期，但由于具体的分子机制尚不明确，且缺乏有效的预警分子和干预靶点，极大制约了对糖尿病血管病变的有效防治。外泌体（EXs）是细胞间信息交流和功能调控的重要手段，亦参与了血管内皮功能的损伤及修复过程。本研究采用细胞和分子生物学、miRNA array、生物信息学、基因敲除及疾病动物模型及临床研究等多学科交叉的新方法，以期阐明在糖尿病患者体内聚集的晚期糖基化终产物（advanced glycation end-products，AGEs）是否通过循环单核巨噬细胞所分泌外泌体（M-EXs）影响血管内皮功能及血管病变进展。研究发现：在AGEs诱导M-EXs携带的miR-22水平发生显著改变，Mir-22下调HCAECs的增殖及成管能力，过表达Lenti-anti-Mir-22的HCAECs VEGFR蛋白表达增加，证实了在HCAECs中VEGFR是Mir-22的靶基因。通过Rab27a KO糖尿病模型建立，我们发现基因敲除模型中外泌体的数量明显下降，Rab27a KO与WT老鼠比较，心功能下降较为明显；且内皮细胞成管能力明显下降。临床研究部分发现：糖尿病患者EVs对ACH依赖的舒张血管能力明显下调，在抑制eNOS情况下，糖尿病患者EVs舒张血管能力下降更加明显。此外，我们还对低氧预处理诱导外泌体变化及其对内皮功能研究做了初步探讨，目前研究表明：低氧预处理hucMSC分泌的外泌体可增强HUVEC增殖、迁移和小管形成能力。基于研究结果初步证实：不同因素导致的外泌体变化可影响内皮细胞增殖、迁移和小管形成能力，AGEs引起循环单核巨噬细胞相关外泌体数量及携带miRs水平改变，并将信号传递到ECs，通过调控其靶基因导致内皮功能紊乱，参与糖尿病血管病变加速进展。研究结果可为糖尿病血管病变防治提供新的靶点。