Tumor necrosis factor α primes cerebral endothelial cells for erythropoietin-induced angiogenesis

Erythropoietin (EPO) enhances angiogenesis in the ischemic brain. Stroke induces secretion of tumor necrosis factor alpha (TNF-alpha). We investigated the effect of TNF-alpha on EPO-induced in vitro angiogenesis in cerebral endothelial cells. Using a capillary-like tubular formation assay, we found that transient incubation of primary rat cerebral microvascular endothelial cells (RECs) with TNF-alpha substantially upregulated EPO receptor (EPOR) expression and addition of EPO into TNF-alpha-treated RECs significantly augmented the capillary-like tube formation. Blockage of TNF receptor 1 (TNFR1) suppressed TNF-alpha-upregulated EPOR expression and abolished EPO-induced tube formation. Attenuation of endogenous EPOR with small interfering RNA (siRNA) also inhibited EPO-enhanced tube formation. Treatment of RECs with EPO activated nuclear factor-kappa B (NF-kappa B) and Akt. Incubation of the TNF-alpha-treated endothelial cells with EPO activated vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), angiopoietin 1 (Ang1), and Tie2. Blockage of VEGFR2 and Tie2 resulted in reduction of EPO-augmented tube formation. These data indicate that interaction of TNF-alpha with TNFR1 sensitizes cerebral endothelial cells for EPO-induced angiogenesis by upregulation of EPOR, which amplifies the effect of EPO on activation of the VEGF/VEGFR2 and Ang1/Tie2 pathways. Our results provide the evidence for crosslink between TNF and EPOR to coordinate the onset of angiogenesis in cerebral endothelial cells. Journal of Cerebral Blood Flow & Metabolism (2011) 31, 640-647; doi: 10.1038/jcbfm.2010.138; published online 11 August 2010