CONICET | Buscador de Institutos y Recursos Humanos

Background: In addition to its well-known hematopoietic effects, erythropoietin (Epo) also has neuroprotective properties. However, hematopoietic side effects are unwanted for neuroprotection, underlining the need for Epo-like compounds with more selective neuroprotective action. One such compound extensively assayed is the chemically modified Epo-derivative carbamylated erythropoietin (cEpo), which in experimental assays cEpo has demonstrated non-hematopoietic tissue protection without significant erythropoietic effect. However, less is known about cellular mechanisms of action. Aims: To compare the neuroprotective action between Epo and cEpo and to distinguish their effects upon erythropoietic cells. Methods: Cell viability/proliferation was analyzed by the MTT assay and apoptosis by fluorescent microscopy after Hoechst staining. Models: Cells of neuronal origin: SH-SY5Y cells were cultured in E-MEM:Ham-FBS. Cells of ability to erythroid differentiation: a) Epo-dependent UT-7 cells were cultured in IMDM-FBS with Epo, b) Epo-independent K562 cells were grown in RPMI-1640-FBS and differentiated to erythroid lineage with hemin, and c) physiological colony forming units-erythroid (CFU-E) from Balb/c mice were evaluated by bone marrow cell cultures in methylcellulose-medium. After 48 h, 2,7-diaminofluorene reaction was performed and hemoglobinized colonies were counted. Results: Epo and cEpo acted in similar way (no significant differences) to prevent apoptosis induced by either staurosporine (STP) or TNF-alpha in SH-SY5Y cells (Control 8±1.5%; STP 44±5.2%; Epo-STP 12±2.2%; cEpo-STP 12±1.7%; TNF 32±1.7%; Epo-TNF 9±0.9%; cEpo-TNF 10±2.9%; n=4). Moreover, assays in the presence of inhibitors showed that cell activation by Epo and cEpo involves similar signaling pathways mediated by PI3K and Jak2. Instead, Epo but not cEpo overcame apoptosis induced by TNF-alpha in erythroid differentiated K562 cells (Control 8±2.3%; TNF 17±2.7%; Epo-TNF 8±1.0%; cEpo-TNF 18±1.5%, Epo-TNF vs. Epo P<0.05, n=4). Different from Epo, cEpo did not prevent apoptosis in cultures of UT-7 cells (Without Epo 39±5.8%; Epo 4±0.3%; cEpo 34±6.2%, n=4). Besides, Epo but not cEpo acted as growth factor in the development of mice CFU-E (Figure). To investigate probable competition between Epo and its carbamylated derivative, cEpo dose-response analysis were performed in the presence of Epo. Results showed that CFU-E growth in the presence of equal amounts of Epo and cEpo (20 ng) did not change the effect of Epo alone. Interestingly, higher amounts of cEpo (200 ng) inhibited the effect of Epo (20 ng) when both compounds were added simultaneously to cultures, but this inhibitory effect disappeared when cEpo was added 60 min after cellular stimulation by Epo (Figure: * Significant differences vs. [Without Epo] or [cEpo] or [Epo+cEpox10], P<0.001, n=10). Conclusions: This work adds new information about the neuroprotective and non-erythropoietic functions of cEpo. Similar mechanisms of action as anti-apoptotic factors in neuronal cells may be suggested for both compounds. However, results showing that the interference of high level of cEpo only occurred at early stages during the interaction of Epo with its erythroid target cell arose for the first time the possibility that cEpo block or modulate the activation of the Epo receptor. This finding may become crucial because it has been reported that relatively high doses of cEpo are needed to act as a neuroprotective factor.

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