| Summary: | Failure of surgical or endo-vascular treatment in critical limb ischemia (CLI) often leads to amputation of leg. Erythropoietin (EPO) has tissue-protective properties, which can be utilized for managing CLI. However EPO also causes haemopoiesis that precludes its use in CLI. Non-haematopoietic EPO derivatives including ARA 290 act specifically on EPO’s tissue-protective receptor (EPOR-CD131) and avoid the haematopoetic side-effects. The aims of this study were to demonstrate the expression of EPOR-CD131 in CLI; develop a model system to simulate myotube ischaemia in vitro; demonstrate the anti-apoptotic and anti-inflammatory potential of EPO and ARA- 290 in vitro; and assess the proangiogenic properties of EPO and ARA 290 in vitro. Samples were obtained from gastrocnemius muscle of patients undergoing leg amputation for CLI (n=12). Controls were obtained from patients undergoing heart surgery (n=12). EPOR-CD131 expression was demonstrated by immunohistochemistry and western blot. An in vitro model of myotube ischemia was developed and myotubes were subjected to simulated-ischemia after pre-treatment with EPO or ARA 290. Apoptosis was measured by nuclear staining, cleaved caspase-3 and LDH-release assays. Inflammatory cytokines were measured by ELISA. Angiogenic potential of EPO and ARA-290 was assessed in HMEC-1 by proliferation, migration and capillary-like tube formation assays. There was clear expression and colocalization of EPOR-CD131. The expression was upregulated in CLI (p < 0.01). ARA-290 and EPO significantly decreased the number of apoptotic nuclei, cleaved caspase-3, LDH and Interleukin-6 release in myotubes exposed to simulated ischemia (p<0.01). However, only EPO significantly increased proliferation, migration and capillary-like tube formation of HMEC-1 (p < 0.05). This is the first study demonstrating expression of EPO receptors within skeletal muscle and their elevated expression in CLI. Using a model for simulated ischaemia of myotubes, it was shown that EPO and ARA 290 decrease inflammation and apoptosis of ischemic myotubes. The use of EPO derivatives to selectively enhance the tissue protective activity of EPO may provide a novel therapeutic avenue for CLI.
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