RECOMBINANT-HUMAN-ERYTHROPOIETIN STIMULATES ANGIOGENESIS IN-VITRO

Endothelial cell migration and proliferation are the key steps in the angiogenic process, and both are stimulated by recombinant human eryth ropoietin (rHuEPO). In addition rHuEPO can increase endothelin-1 (ET-1 ) release by the endothelial cell. We designed the present study to ad dress the question of whether rHuEPO stimulates angiogenesis. An in vi tro quantitative assay for angiogenesis was used. This consisted of ra t aortic rings embedded in a reconstituted basement membrane matrix an d incubated with and without rHuEPO for eight days. We found that rHuE PO 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 significant ly 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 r HuEPO-induced angiogenesis, rings were exposed to ET-1 alone (10(-8) M ). We observed an increase in microvessel formation compared to contro l (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 rHu EPO and anti-ET-1 IgG antibody. Stimulation of angiogenesis by rHuEPO was blunted by the ET-1 antibody. We conclude that rHuEPO stimulates a ngiogenesis in vitro, and this effect is due at least in part to the e nhanced autocrine release of ET-1 by rHuEPO.