Abstract 130: Exosomal Cellular Communication in Peripheral Arterial Disease

Background: Exosomes are nano-meter sized vesicles that play a role in cellular communication. Their effects on target cells are influenced by their cellular source and associated niche. We hypothesize that exosomal communication is involved in the local and systemic pathology of peripheral arterial disease (PAD), where exosomes derived from ischaemic muscle contribute to impaired muscle regeneration and endothelial dysfunction. This study aims to investigate the effect of ischaemia on skeletal muscle exosomes and their potential role in PAD. Methods: Exosomes were isolated from C2C12 myoblasts (MB), MTs and ischaemic MTs (IMTs) through ultracentrifugation. Comparative analysis of MT and IMT exosomes was performed using Nanosight, western blotting (WB) and electron microscopy (EM). PKH26 labelled MB, MT and IMT exosomes were co-cultured with myoblasts or human umbilical vein endothelial cells (HUVECs) for 6, 12 and 24hrs. Exosome uptake was assessed by fluorescent microscopy and FACs. The effect of IMT exosomes on myoblast differentiation was assessed by WB for myogenin and myosin heavy chain. Results: Exosomes were secreted by MTs and IMTs at similar frequencies and were structurally similar. Both were positive for exosomal markers CD81 and Alix. Following 24hr co-culture of PKH26 labelled exosomes with myoblasts or HUVECs, over 80% (over 4000 of 5000) of cells fluoresced positively, signifying internalisation of the exosomes by myoblasts and HUVECs. IMT but not MT derived exosomes impaired myoblast differentiation. Conclusion: MT derived exosomes are taken up by both myoblasts and endothelial cells. Whilst ischaemia does not significantly alter MT derived exosomes quantitatively or structurally, internalisation of IMT exosomes impairs differentiation of myoblasts to myotubes. This suggests that in PAD, altered exosomal message secondary to ischaemia, contributes to muscle dysfunction. Our data also indicates that there is muscle-endothelial cell cross-talk via exosomes, which may also contribute to systemic cardiovascular morbidity. Clarification of the functional effects and delineation of the mechanisms involved may lead to new targets that can be manipulated to improve outcomes of patients with PAD.