Abstract
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 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. 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.
Highlights
During past decades, because of increasing aging population and changes in people’s diets, the worldwide incidence of diabetes has increased rapidly
We found that the cultured cells express CD73, CD90, and CD105, which are mesenchymal stem cell markers, but not CD34 and CD45 that stand for hematopoietic cell markers (Fig. 1A)
We selected three inflammatory cytokines (VCAM-1, tumor necrosis factor (TNF)-α, interleukin 6 (IL-6)) to test how the inflammatory factors affect exosomes from morphological aspects using transmission electron microscopy (TEM), and concentration of Mesenchymal stem cells (MSCs)-exo was determined by Nanoparticle tracking analysis (NTA) as well
Summary
Because of increasing aging population and changes in people’s diets, the worldwide incidence of diabetes has increased rapidly. There has more than 420 million of diabetes patients worldwide according to WHO report.[1] Diabetes can lead to various fatal diseases. Diabetic foot, caused by ischemic disease of lower extremities of diabetic patients, can lead to amputation and even death eventually. It has been reported that 15% of diabetic patients developed diabetic foot.[2] The amputation rate of diabetic foot was about 25% − 50%, which is 40 times higher than that of non-diabetic population[3]. The 5-year survival rate of the patients with diabetic foot was only about 50%.[3]. Diabetic foot is caused by ischemic disease of lower extremities of diabetic patients, and the effective therapy is very limited. Exosomes 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
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