Figure 4. In vitro activation of stromal cells by NE.

HDFs (A) and MAFs (B) were serum starved for 24 h and then incubated in the presence or absence of NE (10 μM) alone and in combination with β-ARs antagonist ICI118-551 (1 μM), SR59230A (10 μM) and propranolol (1 μM) for additional 24 h, then αSMA levels were revealed in total lysates by immunoblotting. Anti-actin antibody was used to ensure equal protein loading. (C) MSCs were serum starved for 24 h and then incubated in the presence or absence of NE (10 μM) for additional 24 h. αSMA and VEGFR-2 levels were analyzed by immunoblotting. Anti-actin immunoblot was used to ensure equal protein loading. (D) MSCs were serum starved for 24 h, seeded in Matrigel-coated dishes and incubated with CM from HDFs, treated for 24 h in the presence or absence of NE (10 μM) and/or βARs antagonists ICI118-551 (1 μM), SR59230A (10 μM), **P < 0.001 vs NE stimulated cells. After 24 h formed capillary were photographed and the number of junctions were counted in 6 fields. Pictures are representative of three independent experiments. (E) HDFs were serum starved for 24 h and then incubated with NE (10 μM) for additional 24 h. IL-6, IL-8, VEGF-A and FGF2 transcripts were evaluated by quantitative Real-Time PCR as described in Material and Methods. Data represent the mean of three independent experiments and are shown as fold change compared to untreated HDFs, *P < 0.05, **P < 0.001. (F) A375 cells were serum starved for 24 h and then incubated in the presence or absence of IL-6 (50 ng/ml), IL-8 (50 ng/ml), VEGF-A (50 ng/ml) and FGF-2 (50 ng/ml) for additional 24 h. β3 and β2 AR expression was evaluated in total lysates by immunoblotting. Anti-actin immunoblot was used to ensure equal protein loading. Figure is representative of three independent experiments.