Effects of erythropoietin on astrocytes and brain endothelial cells in primary culture during anoxia depend on simultaneous signaling by other cytokines and on duration of anoxia

Studies on animals revealed neuroprotective effects of exogenously applied erythropoietin (EPO) during cerebral ischemia/hypoxia. Yet, application of exogenous EPO in stroke patients often lead to haemorrhagic transformation. To clarify potential mechanism of this adverse effect we explored effects of EPO on viabilities of astrocytes and brain endothelial cells (BECs) in primary culture during anoxia of various durations, in the presence or absence of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang1), which are cytokines that are also released from the neurovascular unit during hypoxia. Anoxia (2-48 h) exerted marginal effects on BECs' viability and significant reductions in viability of astrocytes. Astrocyte-conditioned medium did not exert effects and exerted detrimental effects on BECs during 2 h and 24 h anoxia, respectively. This was partially reversed by inhibition of Janus kinase (Jak)2/signal transducer and activator of transcription (STAT)5 activation. Addition of rat recombinant EPO (rrEPO) during 2 h-6h anoxia was protective for astrocytes, but had no effect on BECs. Addition of rrEPO significantly reduced viability of BECs and astrocytes after 48 h anoxia and after 24 h-48 h anoxia, respectively, which was attenuated by inhibition of Jak2/STAT5 activation. Simultaneous addition of rrEPO and VEGFA (1-165) caused marginal effects on BECs, but a highly significant protective effects on astrocytes during 24-48 h anoxia, which were attenuated by inhibition of Jak2/STAT5 activation. Simultaneous addition of EPO, VEGFA 1-165 and Ang1 exerted protective effects on BECs during 24 h-48 h anoxia, which were attenuated by addition of soluble Tie2 receptor. These data revealed that EPO could exert protective, but also injurious effects on BECs and astrocytes during anoxia, which depended on the duration of anoxia and on simultaneous signaling by VEGF and Ang1. If these injurious effects occur in stroke patients, they could enhance vascular damage and haemorrhagic transformation. (C) 2017 Elsevier Ltd. All rights reserved.