Figure 3.

Androgens inhibit while hypoxia induces angiogenic activity in normal prostate stromal cells. Normal prostate stromal cells (PrSC) were treated with DHT (0-10⁻⁷ M) or grown under standard conditions (normoxia) or hypoxia-treated conditions [50-100 μM CoCl₂ or grown in a hypoxia chamber, 0.5% O₂ (hypoxia)] and serum-free conditioned media collected. (a) Angiogenic activity was assessed in conditioned media (20 μg/ml) using a microvascular endothelial cell migration assay. Data are presented as the mean ± SE of the percent maximum migration toward the positive control. (b) VEGF levels were assayed by ELISA. Data are presented as mean ± SD of two replicates. (c) TSP-1 expression was evaluated by Western blot. The lower panel is an identically loaded Coomassie-stained gel to show equal protein loading between lanes. Relative intensity was determined by densitometry. Protein size standard is indicated. Angiogenic activity (d) and VEGF levels (e) were measured as above in hypoxia-treated cells. (f) TSP-1 expression was evaluated as above. (g) The functional activity of TSP-1 in media conditioned by PrSC treated with DHT (10⁻⁷ M) and/or CoCl₂ (100 μM) was tested by the addition of neutralizing anti-TSP-1 antibody in a migration assay. Data are presented as the mean ± SE of the percent maximal migration toward the positive control. *Significantly different compared to untreated controls in each experiment, P<0.05. **Samples between bars are significantly different, P<0.05. ^ΨSignificantly increased compared to untreated or DHT-treated, P<0.002, but not significantly different than CoCl₂ alone, P=0.64. ^πSignificantly increased over the combined hypoxia and DHT treatment sample, P<0.00001.