Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia.
OBJECTIVE
Managing critical limb ischemia (CLI) is challenging. Furthermore, ischemic myopathy prevents good functional outcome after revascularization. Hence, we have focused on limiting the tissue damage rather than angiogenesis, which has traditionally been the motivation to develop nonsurgical treatments for CLI. Erythropoietin (EPO) protects ischemic tissue, and this property may also benefit CLI. The objective of this study was to examine the expression of the tissue-protective EPO receptor complex (EPOR-CD131 [β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor]) in skeletal muscle obtained from humans with CLI. Because native EPO is thrombogenic, the antiapoptotic and anti-inflammatory effects of a nonhematopoietic helix-B peptide of EPO (ARA 290) were investigated on ischemic myotubes in vitro.


METHODS
Tissue was obtained from gastrocnemius muscle of 12 patients undergoing amputation for CLI and from 12 patients without limb ischemia. The expression of EPOR and CD131 was demonstrated by immunohistochemistry and Western blot. A validated in vitro model of myotube ischemia was used in which mature C2C12 myotubes were cultured 6 to 12 hours in a depleted media and gas mixture (20% CO2 and 80% N2). The myotubes were pretreated with EPO or ARA 290 before exposure to simulated ischemia. Apoptosis and cell death were determined by cleaved caspase-3 assay and lactate dehydrogenase release assay. Enzyme-linked immunosorbent assay measured the inflammatory cytokines.


RESULTS
EPOR and CD131 were expressed and significantly upregulated in CLI (average optical density [OD] in Western blot [control vs CLI] EPOR, 0.05 U vs 0.1 U; CD131, 0.10 U vs 0.22 U; P < .01). There was colocalization of EPOR and CD131 in the sarcolemma (cell membrane) of the skeletal myofiber. There was no difference in the distribution of colocalization between the CLI and the normal muscle. The ischemic myotubes treated by ARA 290 in vitro had a significantly decreased number of apoptotic cells (ischemia vs ischemia plus ARA 290: 71.1% vs 55.1%; P < .01), cleaved caspase-3 (OD of ischemia vs ischemia plus ARA 290: 0.15 U vs 0.02 U; P < .01), lactate dehydrogenase release (ischemia vs ischemia plus ARA 290: 32.5 U/L vs 21.3 U/L; P < .01), and IL-6 release (OD at 450 nm, ischemia vs ischemia plus ARA 290: 0.18 vs 0.13; P < .01).


CONCLUSIONS
This study demonstrates the expression and the upregulation of EPOR and CD131 in CLI and also shows that EPOR and CDI are colocalized in the cell membrane of both ischemic and control muscle fiber. The in vitro experiments demonstrate that ARA 290 decreases inflammation and apoptosis of ischemic myotubes. ARA 290 may potentially be used as adjunctive treatment for CLI.
Royal Free Vascular Unit, Division of Surgery & Interventional Science, University College London, London, United Kingdom.
Dhiraj
D
Joshi
Centre for Rheumatology and Connective Tissue Disease, University College London, London, United Kingdom.
David
D
Abraham
Centre for Rheumatology and Connective Tissue Disease, University College London, London, United Kingdom.
Xu
X
Shiwen
Royal Free Vascular Unit, Division of Surgery & Interventional Science, University College London, London, United Kingdom.
Daryl
D
Baker
Royal Free Vascular Unit, Division of Surgery & Interventional Science, University College London, London, United Kingdom. Electronic address: janice.tsui@ucl.ac.uk.
Janice
J
Tsui
D000368:Aged; D017209:Apoptosis / Q000187:drug effects; D016022:Case-Control Studies; D053148:Caspase 3 / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D053648:Cytokine Receptor Common beta Subunit / Q000032:analysis / Q000378:metabolism*; D004921:Erythropoietin / Q000494:pharmacology; D005121:Extremities / Q000098:blood supply; D005260:Female; D006801:Humans; D007249:Inflammation / Q000188:drug therapy / Q000378:metabolism; D015850:Interleukin-6 / Q000378:metabolism; D007511:Ischemia / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D017467:Receptors, Erythropoietin / Q000032:analysis / Q000378:metabolism*; D015854:Up-Regulation
D053648:Cytokine Receptor Common beta Subunit; D015850:Interleukin-6; D009842:Oligopeptides; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; C576178:cibinetide; D007770:L-Lactate Dehydrogenase; D053148:Caspase 3