Carbamylated darbepoetin derivative prevents endothelial progenitor cell damage with no effect on angiogenesis

Abstract 

Erythropoietin (EPO) prevents cell apoptosis induced by oxidative stress. Carbamylated EPO maintains the tissue-protective activities of the unmodified EPO but does not stimulate erythropoiesis. This study evaluates whether carbamylated erythropoietin is as effective as recombinant human erythropoietin in protecting endothelial progenitor cells (EPCs) from apoptosis without stimulating erythropoiesis. Experiments were performed in an erythroid cell line (UT-7) and in human EPCs. Cell signals regulating proliferation and apoptosis (Jak-2, Akt, Erk1/2, NFκB and Stat-5) were measured by Western blotting. In human EPCs, cell senescence, apoptosis and proliferation were assessed by acidic β-gal and measurement of telomere length, TUNEL and PCNA labeling, respectively. Angiogenesis was evaluated using the endothelial tube formation assay. In UT-7, carbamylated erythropoietin (C-darbe) induced phosphorylation of the anti-apoptotic Jak-2/Akt signal and, as opposed to recombinant human erythropoietin (darbe), did not produce a significant activation of cell proliferating signals. Darbe increased the percent of proliferating EPCs and promoted angiogenesis. By contrast, C-darbe failed to stimulate proliferation of EPCs. Both C-darbe and darbe equally reduced apoptosis and senescence. Thus, C-darbe protects EPCs from apoptosis and does not increase erythropoiesis.

Abbreviations: EPCs, endothelial progenitor cells, EPO, erythropoietin, CKD, chronic kidney disease, rHuEPO, recombinant human erythropoietin, darbe, darbepoetin, C-darbe, carbamylated darbepoetin, TNBSA, trinitrobenzenesulfonic acid, PCNA, proliferating cell nuclear antigen, KMESF, molecular equivalents of soluble fluorochrome units

Keywords: Angiogenesis, Apoptosis, Carbamylated darbepoetin, Cell senescence, Endothelial progenitor cells, Inflammation, Proliferation