A third of patients with critical limb ischaemia (CLI) eventually require amputation. Inconsistencies between successful revascularisation and functional outcomes exist, and underlying musculopathy in CLI patients has been identified. Erythropoietin (EPO) has tissue-protective effects in response to ischaemic injury, but its clinical use is often precluded by thromboembolic side effects. Non-haematopoietic EPO-derivatives have been designed to retain only tissueprotective functions of EPO. We hypothesised that ARA-290 (EPO-derivative) may have tissue-protective potential that could represent a novel therapeutic adjunct in patients with CLI. The effect of EPO and ARA-290 in mediating cytoprotection in an in vitro simulated ischaemia model of skeletal muscle was assessed firstly in the immortalised murine C2C12 myoblast cell line and subsequently in skeletal myoblasts isolated from CLI and control donors. In human and murine cells, simulated ischaemia alone demonstrated a detrimental effect on cell function and survival. Addition of EPO or ARA-290 demonstrated significant improvements in function and survival and utilised JAK2/STAT3, PI3k/Akt and NF!B signalling molecules. Isolation of human skeletal myoblasts from CLI patients has not previously been described. Comparison of CLI and control myoblasts elucidated significant differences in their function and survival under both normoxic and simulated ischaemic conditions. CLI myoblasts and myotubes exhibited increased proliferative capacity but reduced migratory and contractile function and importantly a reduced susceptibility to a second ischaemic-insult compared with control myoblasts and myotubes. Evaluation of several variations in the hindlimb ischaemia model allowed the creation of a model which closely recapitulated the muscular pathology observed in human CLI patients. ARA-290 demonstrated improved functional, histological and perfusion outcomes compared to EPO or vehicle-control treated animals. These studies demonstrate the potential of ARA-290 to protect tissues and cells from ischaemic-injury and encourages the development of novel pharmacological therapies for use in patients with “no option” CLI or severe functional deficit.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:634696 |
| Date | January 2014 |
| Creators | Yu, S. R. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1458006/ |
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