Guangdong Key Laboratory of Age-Related Cardiac and Cerebral Diseases, Institute of Neurology, Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China.
Institute of Biochemistry and Molecular Biology, Guangdong Medical University, Zhanjiang, China.
Biol Res. 2023 Apr 1;56(1):16. doi: 10.1186/s40659-023-00418-5.
BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXs) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo.
EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXs. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXs were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXs. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis.
In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXs was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXs could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXs were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXs could be abolished by a PI3K inhibitor (LY294002).
Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.
背景/目的：糖尿病（DM）极易发生糖尿病下肢缺血（DHI）。miR-17-5p 在 DM 中下调，在血管保护中发挥关键作用。内皮祖细胞（EPC）释放的外泌体（EPC-EXs）通过将其内含物 miR 转移至靶细胞，有助于血管保护和缺血组织修复。在此，我们研究了富含 miR-17-5p 的 EPC-EXs（EPC-EXs）是否在体外和体内对 DHI 中的血管和骨骼肌具有显著的保护作用。
转染 scrambled 对照或 miR-17-5p 模拟物的 EPC 用于生成 EPC-EXs 和 EPC-EXs。Db/db 小鼠进行下肢缺血手术。手术后，EPC-EXs 和 EPC-EXs 每 7 天一次注射到下肢腓肠肌，共 3 周。评估下肢血流、微血管密度、毛细血管血管生成、腓肠肌重量、结构完整性和凋亡情况。将血管内皮细胞（ECs）和肌母细胞（C2C12 细胞）置于缺氧加高葡萄糖（HG）条件下，并与 EPC-EXs 和 EPC-EXs 共培养。使用生物信息学分析 miR-17-5p 的潜在靶基因，测量 SPRED1、PI3K、磷酸化 Akt、cleaved caspase-9 和 cleaved caspase-3 的水平，并使用 PI3K 抑制剂（LY294002）进行通路分析。
在 DHI 小鼠模型中，miR-17-5p 在下肢血管和肌肉组织中明显降低，而 EPC-EXs 输注比 EPC-EXs 更能有效增加 miR-17-5p 水平、血流、微血管密度和毛细血管血管生成，并促进腓肠肌重量、产力和结构完整性，同时减少肌肉凋亡。在缺氧加 HG 损伤的 ECs 和 C2C12 细胞中，我们发现 EPC-EXs 可以将其携带的 miR-17-5p 递送到靶 ECs 和 C2C12 细胞，并随后下调靶蛋白 SPRED1，同时增加 PI3K 和磷酸化 Akt 的水平。EPC-EXs 比 EPC-EXs 更能有效降低缺氧加 HG 损伤的 ECs 的凋亡和坏死，同时增加细胞活力、迁移和管状形成，降低 C2C12 细胞的凋亡，同时增加细胞活力和肌管形成。这些 EPC-EXs 的作用可以被 PI3K 抑制剂（LY294002）所阻断。
我们的结果表明，miR-17-5p 通过保护血管内皮细胞和肌细胞功能，促进 EPC-EXs 对 DHI 的有益作用。
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