Abstract

Background
Little is known about the pathological crosstalk between the skeletal muscle and vascular network in Peripheral Artery Disease (PAD). Exosomes are nano-sized vesicles that shuttle microRNA signalling messages between cells, with evidence that the micro-environment can alter their miRNA content.

Methods
Exosomes were isolated from normoxic C2C12 myotubes (MTnorm) and myotubes exposed to simulated ischaemia (MThyp) through ultracentrifugation. Exosomes were then co-cultured with human umbilical vein endothelial cells (HUVECs) and uptake assessed through fluorescence image analysis and flow cytometry. Aortic ring and angiogenesis assays were used to determine the angiogenic potential of MThyp exosomes. RNA Sequencing was used to identify significantly differentially expressed miRNA in MThyp exosomes followed by in silico enrichment analysis using Ingenuity Pathway Analysis software to determine which pathways are targeted.

Results
Exosomes secreted by MTnorm and MThyp cells were taken up by over 80% HUVECs in 24 h. MThyp exosomes significantly increase HUVEC tube length (P = 0.037) compared to MTnorm exosomes and number of loops (P = 0.012) in the tube formation and aortic ring assays. RNA-Sequencing revealed 30 significantly upregulated miRNA in MThyp exosomes, of which, miRNA-181d was identified as a promoter of angiogenesis through inhibition of CDKN1B and TIMP3.

Discussion
MThyp exosomes are taken up by endothelial cells and have proangiogenic properties compared with MTnorm exosomes through overexpression of miRNA-181d. The contribution of this muscle-endothelial cell crosstalk in the pathogenesis of PAD requires further investigation.