Production of sVEGFR-1 by mononuclear phagocytes increases with decreasing O2 concentration. (A) Human peripheral blood monocytes were cultured at 21% (ambient) O2, 5% O2 (mild hypoxia), or 0.5% O2 (severe hypoxia) in media alone or media containing 100 ng/mL GM-CSF. After 48 hours, culture supernatants were harvested and analyzed for sVEGFR-1 content by ELISA. Data are mean ± SEM of 7 normal donors. (B) Monocytes were stimulated with GM-CSF at ambient O2 or at 0.5% O2. Culture supernatants were harvested at the indicated time points and analyzed for sVEGFR-1 content by ELISA. Data are the mean ± SEM of 6 normal donors. (C) Monocytes were stimulated as in panel B and analyzed for sVEGFR-1 transcript by real-time PCR at the indicated time points. Data are the mean ± SEM of 6 normal donors. (D) Supernatants from the same cells as in panel A were measured using an ELISA that detects only bioavailable VEGF (ie, VEGF that is not bound to sVEGFR-1). (E) VEGF levels under the same conditions as in panel B. (F) VEGF transcript levels under the same conditions as in panel B. (G) Monocytes were cultured at ambient O2 or 0.5% O2 with increasing concentrations of GM-CSF (0.1 ng/mL to 100 ng/mL). sVEGFR-1 secretion after 48 hours was measured by ELISA. (H) VEGF levels in the same supernatants as in panel G. (I) HUVECs were cultured in Matrigel in unsupplemented media (negative control), media supplemented with 10 ng/mL VEGF (positive control), or supernatants derived from monocytes cultured at 0.5% O2 in the presence or absence of GM-CSF. A representative image from each condition is shown (left). The total number of tubes formed between discrete endothelial cells were counted in each well. Graphed results (right) represent the average ± SEM number of tubes/field using supernatants from 4 different monocyte donors. (J) Peripheral blood monocytes were differentiated into macrophages by 3-day culture with M-CSF before stimulation with 100 ng/mL GM-CSF at either ambient O2 or 0.5% O2. Culture supernatants were harvested after 48 hours, and sVEGFR-1 concentration was measured by ELISA. Data are mean ± SEM of 3 donors. (K) VEGF levels under the same conditions as in panel J. *P < .001 versus all other conditions at the same time point. **P < .05 versus all other conditions at the same time point. Utx indicates untreated.
