Human mesenchymal stem cells promote ischemic repairment and angiogenesis of diabetic foot through exosome miRNA-21-5p

Background: 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. Methods: 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. Results: We found that inflammatory cytokines (tumor necrosis factor a and interleukin-6) and vascular cell adhesion 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 exosomemiRNA-21-5p 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. Conclusions: 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.

Automated summary

In this study, it was found that inflammatory cytokines and vascular cell adhesion molecule-1 induce MSCs to secrete exosomes heterogeneously. By RNA sequencing, a new proangiogenic miRNA, miRNA-21-5p, was defined. Knockdown and overexpression experiments validated the biological activity of exosome miRNA-21-5p, promoting in vitro cell proliferation, in vivo pro-angiogenesis in CAM assay, and tissue injury and repair angiogenesis in diabetic rat models. Exosome miRNA-21-5p was shown to promote angiogenesis through upregulation of VEGFR, and activation of AKT and MAPK pathways. This work suggests miRNA-21-5p as a potential novel mechanism for exosomes to promote ischemic tissue repair and angiogenesis in diabetic foot, with potential implications for clinical therapy.