The NO-stimulatory actions of EPO require VEGF-R2 and its downstream signaling. (A) Bone marrow cells isolated from wild-type C57/BL6 mice and βC-R knockout mice were grown for 1 week under conditions that promoted EPC growth. Then the cells were trypsinized and allowed to plate for 24 hours before treatment with 100 ng/mL VEGF for 3 hours before measurement of bioavailable NO by DAF-FM. Shown are the representative results of 2 independent experiments as mean ± SD (n ≥ 20). *P < .01. (B) CD34⁺ EPCs were isolated from healthy controls and incubated with isotype IgG, 50 mU/mL EPO alone or in combination with 0.01μg of anti-VEGF, anti-VEGF-R2, or isotype IgG 3 hours before measuring bioavailable NO with DAF-FM. Shown are the representative results of 3 independent experiments as mean ± SD (n ≥ 23). *P < .001 compared with untreated. (C) CD34⁺ EPCs were isolated from healthy controls and incubated with VEGF (100 ng/mL) and/or EPO (50 mU/mL) alone and in combination with anti-VEGF-R1 (1 μg/mL) or SU1498 (5μM). Shown are the representative results of 2 independent experiments as mean ± SD (n ≥ 15). *P < .001 compared with control. #P < .001 compared with control. (D) CD34⁺ EPCs were isolated from healthy controls and after 24 hours transfected with control shRNA or VEGF-R2 shRNA. After another 24 hours, the cells were treated with 50 mU/mL cEPO, as indicated, and DAF-FM fluorescence was determined. Shown are the representative results of 2 independent experiments as mean ± SD (n ≥ 10). *P < .001 compared with untreated. (E) CD34⁺ EPCs were isolated from healthy controls and incubated with AG490 (10μM) alone and in combination with EPO (50 mU/mL). Shown are the representative results of 3 different experiments as mean ± SD (n ≥ 32). *P < .001 compared with control. (F) CD34⁺ EPCs were isolated from healthy controls and incubated with LY294002 (5μM) alone and in combination with EPO (50 mU/mL). Shown is a representative of 4 independent experiments as mean ± SD (n ≥ 37). *P < .001 compared with control, no EPO.