Effects of rHuEpo on cellular proliferation and endothelin-1 production in cultured endothelial cells

Background. Although elevation of blood pressure is considered to be the main adverse effect under rHuEpo therapy in haemodialysis patients, the precise mechanism remains obscure. The direct effect of rHuEpo on endothelial cells (EC) has been suggested as one of contributing factors of rHuEpo-induced hypertension. Methods. EC were incubated with various concentrations of rHuEpo (0, 1000, 5000, 10 000 mU/ml) for up to 7 days, and cell numbers, DNA and protein synthesis by EC and supernatant concentrations of immunoreactive endothelin-1 (ET) were determined by haemocytometer, 3 H-thymidine and 3 H-leucine incorporation, and RIA, respectively. The effect of rHuEpo on EC proliferation was confirmed by anti-rHuEpo rabbit antiserum. The effect of cycloheximide or actinomycin D was also examined on the increase in ET production by rHuEpo. Results. rHuEpo dose-dependently stimulated the proliferation of cultured EC, and this proliferative effect was inhibited by anti-rHuEpo rabbit antiserum. DNA and protein syntheses by EC were also increased by rHuEpo. The supernatant concentrations of ET cultured with rHuEpo at 5000 mU/ml or more showed significantly greater values than those without rHuEpo and the increase in ET in the supernatants of media containing 5000 mU/ml rHuEpo was inhibited by incubation with 0.2 μg/ml actinomycin D or 10 μg/ml cycloheximide. Further, rHuEpo increased DNA synthesis by EC which had been cultured in E-BM medium containing 0.5 or 2% FBS for 3h and which were recultured in E-BM medium containing 5% FBS for 15 h. Conclusions. rHuEpo directly stimulates EC proliferation as a competence factor, and it also accelerates endothelin-1 production in association with stimulation of DNA and protein syntheses by EC.