No abstract

Endothelial cell migration and proliferation are the key steps in the angiogenic process, and both are stimulated by recombinant human erythropoietin (rHuEPO). In addition rHuEPO can increase endothelin-1 (ET-1) release by the endothelial cell. We designed the present study to address the question of whether rHuEPO stimulates angiogenesis. An in vitro quantitative assay for angiogenesis was used. This consisted of rat aortic rings embedded in a reconstituted basement membrane matrix and incubated with and without rHuEPO for eight days. We found that rHuEPO increased vessel outgrowth after four days of culture and this was continued for the next four days (rHuEPO vs. control: day 4, 12 +/- 2 vs. 4 +/- 1, P < 0.002 and day 8, 124 +/- 18 vs. 56 +/- 12 P < 0.006). Supernatant endothelin-1 (ET-1) levels, at 24 hours, were significantly higher than controls in the rings incubated with rHuEPO (107 +/- 13 vs. 43 +/- 10 pg/ml, P < 0.003). To investigate the role of ET-1 in rHuEPO-induced angiogenesis, rings were exposed to ET-1 alone (10(-8) M). We observed an increase in microvessel formation compared to control (day 4, 4 +/- 2 vs. 2 +/- 1, P < 0.006, and day 8, 67 +/- 12 vs. 51 +/- 10, P < 0.03). In addition, aortic rings were co-cultured with rHuEPO and anti-ET-1 IgG antibody. Stimulation of angiogenesis by rHuEPO was blunted by the ET-1 antibody.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypertension is a major complication of rHuEPO therapy in hemodialysis (HD) patients. We have previously reported that patients receiving rHuEPO intravenously (i.v.) had higher mean arterial pressure (MAP) and plasma endothelin-1 (ET-1) levels than those in which the hormone was administered subcutaneously (s.c.). To test whether the increased serum ET-1 levels associated with i.v. rHuEPO administration are the result of a direct effect of the hormone on ET-1 release by the endothelial cells (EC), we examined the effects of rHuEPO in vitro. Bovine pulmonary artery endothelial cells (BPAEC) were exposed to doses of rHuEPO of 0.8; 1.6; 3.3 and 6.6 U/ml. A 24 hour-time course showed maximal ET-1 production at 12 hours for all the doses tested. A significant increase in cell proliferation over controls was observed at 24 hours, for all rHuEPO doses, and no correlation was found between ET-1 values and cell proliferation. Inhibition of protein synthesis by cycloheximide (10 micrograms/ml) abolished the stimulation of ET-1 release by rHuEPO. Thrombin (4 U/ml) and angiotensin II (10(-7) M), two potent stimulators of ET-1 release, had additive effects to those of rHuEPO. Specific thrombin and angiotensin II antagonists blocked these additive effects, reducing ET-1 release to the level of rHuEPO stimulation alone. In summary, rHuEPO stimulates vascular EC in culture to increase ET-1 release through an increase in synthesis and in a time dependent fashion. The routes of stimulation seem to differ from other known ET-1 secretogoges. Our data also confirm a significant mitogenic effect of rHuEPO on the endothelial cell.