Epoetin alfa (EPO) and its nonerythropoietic derivative, carbamylerythropoietin (CEPO), have been recently shown, in various animal models, to have protective effects on cardiac and brain tissue. CEPO does not stimulate red blood cell production and therefore is devoid of prothrombotic side effects. Circulating endothelial progenitor cells (EPCs) are hematopoietic stem cells that enter peripheral circulation and play a role in vascular repair during myocardial and cerebral ischemia, with the chemokine stromal-derived factor (SDF-1) being a key regulator of their homing. We studied the EPO and CEPO effects on EPC chemotaxis and intracellular nitric oxide (NO) production. Peripheral mononuclear cells from 11 volunteers were incubated with 20 U/mL EPO and 30 U/mL CEPO for 12 hours, followed by isolation of EPC from EPO-treated and control samples. Chemotaxis assay to 100 nM SDF-1 was performed in a 96-well Neuroprobe chemotaxis chamber for 4.5 hours. Intracellular NO production was measured in real time by laser confocal microscopy with DAF-FM diacetate. Pretreatment with EPO and CEPO resulted in significant increase in EPC chemotaxis to SDF-1 (26.57 ± 10.41% and 30.6 ± 11.09% vs 17.43 ± 5.16% of the control cells migrating). In search for mechanisms of EPO effect on chemotaxis, we evaluated intracellular NO, a known mediator of endothelial cell migration. Pretreatment with EPO and CEPO increased the bioavailabe NO within EPC above baseline (2,172 ± 321.04 and 2,223.68 ± 413.0 vs 1,227.56 ± 590.68). In addition, acute incubation with EPO increased the rate of intracellular NO production threefold (56.15 ± 17.49 vs19.52 ± 20.4 relative fluorescence units per 10 minutes, p = .023). When we partially blocked intracellular NO synthase by l-NAME to bring NO content in EPO-treated EPC to a control level, the migratory ability of these cells also diminished (26.57 ± 10.41% vs 21.2 ± 7.2% of the cells migrating). These results suggest that both EPO and CEPO may play a role in vascular repair by activating intracellular NO production with a resulting increase in EPC migration to the site of ischemic injury. It may in part explain the protective effect of EPO and CEPO on cardiac and brain tissue in a setting of acute ischemia.
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