cibinetide and Ischemia

Retinal ischemia remains a common sight threatening end-point in blinding diseases such as diabetic retinopathy and retinopathy of prematurity. Endothelial colony forming cells (ECFCs) represent a subpopulation of endothelial progenitors with therapeutic utility for promoting reparative angiogenesis in the ischaemic retina. The current study has investigated the potential of enhancing this cell therapy approach by the dampening of the pro-inflammatory milieu typical of ischemic retina. Based on recent findings that ARA290 (cibinetide), a peptide based on the Helix-B domain of erythropoietin (EPO), is anti-inflammatory and tissue-protective, the effect of this peptide on ECFC-mediated vascular regeneration was studied in the ischemic retina.. The effects of ARA290 on pro-survival signaling and function were assessed in ECFC cultures in vitro. Efficacy of ECFC transplantation therapy to promote retinal vascular repair in the presence and absence of ARA290 was studied in the oxygen induced retinopathy (OIR) model of retinal ischemia. The inflammatory cytokine profile and microglial activation were studied as readouts of inflammation.. ARA290 activated pro-survival signaling and enhanced cell viability in response to H. Regulation of the pro-inflammatory milieu of the ischemic retina can be enhanced by ARA290 and may be a useful adjunct to ECFC-based cell therapy for ischemic retinopathies.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Endothelium, Vascular; Erythropoietin; Humans; Infant, Newborn; Ischemia; Mice; Mice, Inbred C57BL; Oligopeptides; Retinal Diseases; Retinal Vessels; Signal Transduction; Vasodilation

Alternate erythropoietin (EPO)-mediated signaling via the EPOR/CD131 heteromeric receptor exerts the tissue-protective actions of EPO in a wide spectrum of injuries, especially ischemic diseases. Circulating endothelial progenitor cells contribute to endothelial repair and post-natal angiogenesis after chronic ischemic injury. This work aims to investigate the effects of ARA290, a specific agonist of EPOR/CD131 complex, on a subpopulation of endothelial progenitor cells named endothelial colony-forming cells (ECFCs) and to characterize its contribution to ECFCs-induced angiogenesis after peripheral ischemia.. ARA290 effects on ECFCs properties were studied using cell cultures in vitro. We injected ARA290 to mice undergoing chronic hindlimb ischemia (CLI) in combination with ECFC transplantation. The homing of transplanted ECFC to ischemic tissue in vivo was assessed by SPECT/CT imaging.. In vitro, ARA290 enhanced the proliferation, migration, and resistance to H2O2-induced apoptosis of ECFCs. After ECFC transplantation to mice with CLI, a single ARA290 injection enhanced the ischemic/non-ischemic ratio of hindlimb blood flow and capillary density after 28 days and the homing of radiolabeled transplanted cells to the ischemic leg 4 h after transplantation. Prior neutralization of platelet-endothelial cell adhesion molecule-1 (CD31) expressed by the transplanted cells inhibited ARA290-induced improvement of homing.. ARA290 induces specific improvement of the biological activity of ECFCs. ARA290 administration in combination with ECFCs has a synergistic effect on post-ischemic angiogenesis in vivo. This potentiation appears to rely, at least in part, on a CD31-dependent increase in homing of the transplanted cells to the ischemic tissue.

Topics: Animals; Apoptosis; Blotting, Western; Cells, Cultured; Cytokine Receptor Common beta Subunit; Endothelial Progenitor Cells; Hindlimb; Humans; Hydrogen Peroxide; Ischemia; Mice; Oligopeptides; Oxidative Stress; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Erythropoietin; Signal Transduction; Single Photon Emission Computed Tomography Computed Tomography

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.. 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.. 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).. 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.

Topics: Aged; Apoptosis; Case-Control Studies; Caspase 3; Cell Membrane; Cytokine Receptor Common beta Subunit; Erythropoietin; Extremities; Female; Humans; Inflammation; Interleukin-6; Ischemia; L-Lactate Dehydrogenase; Male; Middle Aged; Muscle Fibers, Skeletal; Oligopeptides; Receptors, Erythropoietin; Up-Regulation