Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia Dhiraj Joshi, MD, MRCS, David.

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Potential role of erythropoietin receptors and ligands in attenuating apoptosis and inflammation in critical limb ischemia Dhiraj Joshi, MD, MRCS, David Abraham, PhD, Xu Shiwen, PhD, Daryl Baker, FRCS, PhD, Janice Tsui, MD, FRCS Journal of Vascular Surgery Volume 60, Issue 1, Pages 191-201.e2 (July 2014) DOI: 10.1016/j.jvs.2013.06.054 Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 1 The expression of erythropoietin receptor complex (EPOR) and β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor (CD131) in C2C12 myotubes. Representative photomicrographs of C2C12 cells. A, Hematoxylin and eosin staining of myoblasts and mature multinucleated myotubes. B, Expression of EPOR in myotubes (ab10653) using immunofluorescence. C, Expression of CD131 in myotubes using immunofluorescence. The arrows represent the expression along the myotubes. The nuclei were stained with 4,′6-diamidino-2-phenylindole. Anti-EPOR antibody (ab10653) and anti-CD131 antibody (DM3566P) was used for showing the expression of EPOR and CD131, respectively. Texas-Red fluorescent dye (Alexa Fluor 594; Alexa Fluor, Paisley, UK) was used. Original magnification ×40. Journal of Vascular Surgery 2014 60, 191-201.e2DOI: (10.1016/j.jvs.2013.06.054) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 2 The expression of erythropoietin receptor complex (EPOR) and β-chain of interleukin (IL)-3/IL-5/granulocyte macrophage colony-stimulating factor receptor (CD131) and its upregulation in human critical limb ischemia (CLI). Representative photomicrographs show human skeletal muscle sections. A, EPOR immunoreactivity is demonstrated by immunohistochemistry. EPOR is expressed predominantly in the sarcolemma (s) of the control myofiber (m); however, a more generalized EPOR expression is present in CLI. The Western blot shows EPOR expression at 59 kDa and its significant upregulation in CLI. B, CD131 immunoreactivity is demonstrated by immunohistochemistry. Western blot shows CD131 expression at 130 kDa and its significant upregulation in CLI. C, EPOR and CD131 immunofluorescent colocalization is demonstrated in the periphery and close to the nucleus (n) of representative ischemic myofiber (m). This suggests predominant colocalization in the sarcolemma. The nuclei are stained with 4,′6-diamidino-2-phenylindole (DAPI). The arrow represents a site of expression or colocalization. Anti-EPOR antibody (ab10653) was used in immunohistochemistry and Western blot; Jurkat and HuT 78 whole cell lysates were used as positive controls for EPOR and CD131, respectively. β-Actin was used as the loading control in Western blot. Anti-EPOR antibody (Ww12) and anti-CD131 antibody (N-20) were used for immunofluorescence colocalization. Data are represented by mean ± standard deviation (error bars); n = 12 for CLI and n = 12 for control. Two-tailed t-test for independent samples was used for analyses. IgG, Immunoglobulin G; PBS, phosphate-buffered saline. Journal of Vascular Surgery 2014 60, 191-201.e2DOI: (10.1016/j.jvs.2013.06.054) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 3 Pretreatment with nonhematopoietic helix-B peptide of erythropoietin (ARA 290) decreases apoptotic nuclear fragmentation and cleaved caspase-3 release in ischemic myotubes in vitro. A, The dose-response curve shows that ARA 290 at 8 ng/mL of culture media was the optimum dose to decrease the maximum number of apoptotic cells. B, Representative fluorescent (grayscale) photomicrographs show increased bright 4,′6-diamidino-2-phenylindole-stained apoptotic nuclei in the ischemic myotubes compared with control normoxic myotubes. The number of apoptotic nuclei was significantly reduced when myotubes were pretreated with ARA 290 before ischemic exposure. C, Representative Western blot of cleaved caspase-3 assay demonstrates significantly decreased cleaved caspase-3 expression in the cell lysates obtained from C2C12 myotubes exposed to simulated ischemia but pretreated with ARA 290. Jurkat cells treated with cytochrome C were used as positive controls, and myotubes incubated in normoxia were used as positive controls. All data are represented by mean ± standard deviation (error bars), n = 9. Two-way analysis of variance/Tukey honestly significant differences test was used. EPO, Erythropoietin. Journal of Vascular Surgery 2014 60, 191-201.e2DOI: (10.1016/j.jvs.2013.06.054) Copyright © 2014 Society for Vascular Surgery Terms and Conditions

Fig 4 Nonhematopoietic helix-B peptide of erythropoietin (ARA 290) pretreatment decreases lactate dehydrogenase (LDH) and interleukin (IL)-6 release from ischemic myotubes in vitro. A, ARA 290 and erythropoietin (EPO) significantly decreased the total LDH released from myotubes (measured as units per liter of media by calorimetry). B, Enzyme-linked immunosorbent assay results show that EPO and ARA 290 significantly attenuated the release of IL-6 measured in the culture media of C2C12 myotubes after exposure to ischemic simulation. Negative control immunoglobin A (IgG A) and positive controls were antibody specific and provided by the manufacturer in the enzyme-linked immunosorbent assay kit. All data are represented by mean ± standard deviation (error bars), n = 9. Two-way analysis of variance/Tukey honestly significant differences test was used. Journal of Vascular Surgery 2014 60, 191-201.e2DOI: (10.1016/j.jvs.2013.06.054) Copyright © 2014 Society for Vascular Surgery Terms and Conditions