Exposure of endothelial cells to recombinant human erythropoietin induces nitric oxide synthase activity

Background. Anemic patients with chronic renal failure receiving recombinan t human erythropoietin (rHuEPO) therapy frequently develop hypertension thr ough an unknown mechanism. We hypothesize that EPO receptors (EPORs) on end othelial cells (ECs) in various sites of vasculature may mediate the activi ties of nitric oxide synthase (NOS) and/or the release of endothelin-1 (ET- 1), contributing to blood pressure changes. We tested this hypothesis using primary cultures of ECs obtained from human coronary artery (HCAEC), pulmo nary artery (HPAEC), dermis (HDEC), and umbilical vein (HUVEC). Methods. EPORs were measured by I-125-EPO binding. The effect of EPO on EPO R, ET-1, and NOS mRNA levels was assessed by quantitative reverse transcrip tion-polymerase chain reaction. Cellular NOS activity and ET-1 release into the medium was measured by the NOSdetect assay and by radioimmunoassay kit s. Results. Short-term (4 h) treatment with EPO (4 U/mL) did not change the nu mber or affinity of EPOR per cell. Neither were there any changes in the am ount of EPOR, ET-1, and NOS transcripts (cDNA/mu g of mRNA) nor in ET-1 rel ease and NOS activity. In HUVEC only, 24-hour exposure to EPO caused a thre efold increase in NOS transcript. In other cells, EPO treatment for six day s increased NOS activity by twofold to fourfold. Conclusions. We show that upon extended exposure, EPO induces NOS activity but does not affect ET-1 release. These findings indicate that the hyperten sive effect of EPO is not likely to be caused by a direct effect on ECs.