Article Figures & Data
Figures
- Fig. 1.
Effect of 17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on angiogenesis in Matrigel (Becton Dickinson). Matrigel containing fibroblast growth factor-2 (FGF-2; 300 ng/pellet) was injected s.c. in mice. A, mice were treated. i.p. with vehicle or 17-AAG and 17-DMAG (25 mg/kg, daily ×6). B, mice were treated p.o. daily with vehicle or 17-DMAG (50, 25, 12 mg/kg, daily ×6). At day 7, pellets were collected, and the angiogenic response was evaluated by measuring the hemoglobin content of the pellets. Data are expressed as hemoglobin content (g/dl) for each pellet. Horizontal bars, median. ∗, P < 0.0001, ∗∗, P < 0.0002, ∗∗∗, P < 0.04 compared with vehicle-treated, FGF-2-containing pellets (Mann-Whitney U test).
- Fig. 2.
Histological analysis of Matrigel treated with 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG). Experiment was performed as in Fig. 1<$REFLINK> . Negative control (w/o fibroblast growth factor-2; FGF-2): Matrigel containing no angiogenic stimulus showed few infiltrating cells (A) and a weak CD31 immunostaining. B, positive control (FGF-2): Matrigel containing FGF-2 in vehicle-treated mice presented a high degree of cellularity, and the presence of blood-containing vessels (C), confirmed by a high number of CD31 positive vessels (D); with 17-DMAG-treated mice (50 mg/kg, p.o.): the FGF-2-containing Matrigel pellets presented a reduced number of blood-containing vessels (E) and no positivity for CD31 (F). Results show a representative example of histological analysis and CD31 immunostaining. n = 5 mice per group.
- Fig. 3.
17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) induce growth inhibition of endothelial cells. Human umbilical vascular endothelial cells were plated in media devoid of growth factors for 24 h and then stimulated with 10 ng/ml of fibroblast growth factor-2 (A) or 10 ng/ml of vascular endothelial growth factor (B) in the presence of various concentrations of 17-DMAG. Cells were incubated for 4 h (○), 24 h (□), 48 h (▵), and 72 h (⋄). Results are expressed as percentage of control (vehicle-treated cells) and are the mean of triplicate; bars, ±SD. Data are from one experiment representative of four.
- Fig. 4.
17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DAMG) induces apoptosis in human umbilical vascular endothelial cells. To determine the apoptotic population, human umbilical vascular endothelial cells were incubated with 17-DMAG in the presence of fibroblast growth factor-2 10 ng/ml (A) or vascular endothelial growth factor 10 ng/ml (B). At the indicated times, apoptotic cells were determined by Annexin V staining as described in “Materials and Methods.” Data are plotted as percentage of Annexin V-positive population. Results are the mean of triplicate and representative of three independent experiments; bars, ±SD.
- Fig. 5.
Effect of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on endothelial cell migration and invasion. A, endothelial cells were exposed to a different concentration of 17-DMAG for 24 h and then tested in migration assay as described in “Materials and Methods.” Fibroblast growth factor-2 (10 ng/ml; ▪) and vascular endothelial growth factor (10 ng/ml; □) were used as chemoattractants. Results are expressed as percentage of control (vehicle-treated cells) and are the mean of triplicate; bars, ±SD. Data are from one experiment representative of three. B, endothelial cells were exposed to different concentration of 17-DMAG for 24 h and then tested in invasion assay as described. Results are expressed as percentage of control (vehicle-treated cells) and are the mean of triplicate. Data are from one experiment representative of two; bars, ±SD.
- Fig. 6.
17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) pretreatment inhibits cord formation. Endothelial cells were plated on a three-dimensional layer of Matrigel where they aligned, forming cord-like structures. Treatment of human umbilical vascular endothelial cells with vehicle (A) or 17-DMAG 10 nm (B), 25 nm (C), 50 nm (D), 125 nm (E), and 250 nm (F) caused a concentration-dependent inhibition of cord formation as it can be seen by the inhibition of cord junctions (○) and cord length (▵). G, data are expressed as percentage of control (vehicle-treated cells). Data are from one experiment representative of three.
- Fig. 7.
Effect of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) on signaling proteins in endothelial cells. Fibroblast growth factor-2 (50 ng/ml; A) or vascular endothelial growth factor (50 ng/ml; B) -stimulated human umbilical vascular endothelial cells were treated for 4 h and 24 h with 17-DMAG at the indicated doses or vehicle as control. Expression levels of pAKT, AKT, c-Raf-1 heat shock protein (Hsp)70, Hsp90, pERK, and extracellular signal-regulated kinase (ERK) proteins were evaluated by Western blot analysis. Actin was used to demonstrate protein loading (40 μg).
Volume 10, Issue 14
