Guangzhou Panyu Central Hospital, Guangzhou 511400, Guangdong, PR China; Medical Imaging Institute of Panyu, Guangzhou 511400, Guangdong, PR China.
Guangzhou Panyu Central Hospital, Guangzhou 511400, Guangdong, PR China.
Stem Cell Res. 2021 Apr;52:102235. doi: 10.1016/j.scr.2021.102235. Epub 2021 Feb 11.
Diabetic foot is caused by ischemic disease of lower extremities of diabetic patients, and the effective therapy is very limited. Mesenchymal stem cells (MSCs) based cell therapy had been developed into a new treatment strategy for diabetic foot clinically. However, the underlying molecular mechanism remains to be fully addressed. Exosomes (extracellular vesicles) secreted by MSCs may play crucial role in the processes of MSCs mediated inhibition of inflammatory microenvironment as well as pro-angiogenesis of ischemic tissue of diabetic foot.
Exosomes were isolated from MSCs using ultracentrifugation, and further characterized by the nanoparticle tracking analyzer and flow cytometry. Moreover, RNA sequencing, Western Blot, in vitro cell proliferation, in vivo pro-angiogenesis, as well as ischemic repairment of diabetic foot through rat model were performed to evaluate exosome physiological functions.
We found that inflammatory cytokines (tumor necrosis factor α and interleukin-6) and vascularcelladhesion molecule-1 induced MSCs to secrete exosomes heterogeneously, including exosome size and quantity. Through RNA sequencing, we defined a new proangiogenic miRNA, miRNA-21-5p. Further knockdown and overexpression of miRNA-21-5p by manipulating MSCs validated the biological activity of exosome miRNA-21-5p, including in vitro cell proliferation, in vivo pro-angiogenesis in Chick Chorioallantoic Membrane (CAM) assay, and in vivo pro-angiogenesis experiments (tissue injury and repair) in diabetic rat models. Furthermore, we discovered that exosome promoted angiogenesis through upregulations of vascular endothelial growth factor receptor (VEGFR) as well as activations of serine/threonine kinase (AKT) and mitogen-activated protein kinase (MAPK). Together, our work suggested miRNA-21-5p could be a novel mechanism by which exosomes promote ischemic tissue repair and angiogenesis. Meanwhile, miRNA-21-5p could be potentially developed into a new biomarker for exosomes of MSCs to treat diabetic foot.
miRNA-21-5p is a new biomarker and a novel mechanism by which exosomes promote ischemic tissue repair and angiogenesis of diabetic foot. Our work could not only provide new scientific evidences for revealing pro-angiogenesis mechanism of MSCs, but also eventually benefit MSCs-based clinical therapy for diabetic foot of diabetes patients.
糖尿病足是由糖尿病患者下肢缺血性疾病引起的，有效的治疗方法非常有限。基于间充质干细胞（MSCs）的细胞治疗已发展成为糖尿病足的一种新的临床治疗策略。然而，其潜在的分子机制仍有待充分阐明。MSCs 分泌的外泌体（细胞外囊泡）可能在 MSC 介导的抑制炎症微环境以及糖尿病足缺血组织的促血管生成过程中发挥关键作用。
使用超速离心法从 MSCs 中分离出外泌体，并通过纳米颗粒跟踪分析仪和流式细胞术进一步进行表征。此外，通过 RNA 测序、Western blot、体外细胞增殖、体内促血管生成以及通过大鼠模型对糖尿病足的缺血修复来评估外泌体的生理功能。
我们发现炎症细胞因子（肿瘤坏死因子-α和白细胞介素-6）和血管细胞黏附分子-1 诱导 MSCs 不均匀地分泌外泌体，包括外泌体的大小和数量。通过 RNA 测序，我们定义了一种新的促血管生成 miRNA，miRNA-21-5p。进一步通过操纵 MSCs 对 miRNA-21-5p 的敲低和过表达验证了外泌体 miRNA-21-5p 的生物学活性，包括体外细胞增殖、鸡胚绒毛尿囊膜（CAM）实验中的体内促血管生成以及糖尿病大鼠模型中的体内促血管生成实验（组织损伤和修复）。此外，我们发现外泌体通过上调血管内皮生长因子受体（VEGFR）以及激活丝氨酸/苏氨酸激酶（AKT）和丝裂原活化蛋白激酶（MAPK）来促进血管生成。总之，我们的工作表明 miRNA-21-5p 可能是外泌体促进缺血组织修复和血管生成的新机制。同时，miRNA-21-5p 可能被开发为治疗糖尿病足的 MSC 外泌体的新型生物标志物。
miRNA-21-5p 是一种新的生物标志物和机制，通过该机制外泌体促进糖尿病足的缺血组织修复和血管生成。我们的工作不仅为揭示 MSC 促血管生成机制提供了新的科学依据，而且最终有益于基于 MSC 的糖尿病患者糖尿病足的临床治疗。
