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The catalytic domain of endogenous urokinase-type plasminogen activator is required for the mitogenic activity of platelet-derived and basic fibroblast growth factors in human vascular smooth muscle cells

Department of Medicine, Hematology and Oncology, University of Münster, Albert-Schweitzer Str. 33, D-48129 Münster, Germany

View larger version (19K): [in a new window]   Fig. 1. Time-dependent increase in DNA synthesis in cultured human vascular SMC stimulated with PDGF or bFGF. Quiescent cells in 96-well plates were treated with 1% FCS ([UNK]); 1% FCS+10 ng/ml bFGF (); or 1% FCS+20 ng/ml PDGF (). Cells were exposed to 4x12 hour BrdU pulses (10 µM) from 0-48 hours in the continuous presence of the growth factors. BrdU uptake was determined by ELISA. Data points represent absorbance levels (medians and IQR, n=6).

View larger version (31K): [in a new window]   Fig. 2. Effect of PDGF and bFGF on UPA expression in human vascular SMC after 24 and 48 hours stimulation. Growth-arrested subconfluent SMC were cultured in DMEM/Ham's F-12 with 0.2% FCS or with 1% FCS in the absence or presence of PDGF (20 ng/ml) and bFGF (10 ng/ml). (A) UPA antigen levels in cell lysates. Data from a minimum of six independent experiments performed in triplicates are given as box-and-whisker plots; boxes denote interquartile ranges with median values shown as horizontal lines inside the boxes. Whiskers denote ranges; outliers beyond 1.5-times interquartile ranges are represented by individual symbols ([UNK]). UPA antigen levels in PDGF- or bFGF-stimulated cells were significantly higher than in cells treated with 1% FCS alone both after 24 hours (P=0.03 for PDGF and P=0.001 for bFGF; Mann-Whitney test) and 48 hours of stimulation (P<0.001 for PDGF and P<0.001 for bFGF; Mann-Whitney test). (B) UPA transcripts in unstimulated (1% FCS) and PDGF- or bFGF-stimulated SMC were analyzed by RT-PCR after 24 and 48 hours treatment. Levels of UPA messenger RNA were estimated by normalization against co-amplified glyceraldehyde-3-phosphate dehydrogenase (GAPDH); M, size marker.

View larger version (22K): [in a new window]   Fig. 3. UPA-mediated DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. Growth arrested cells were exposed to PDGF or bFGF (for 48 hours) in the absence or presence of polyclonal goat anti-UPA IgG (anti-UPA; 40 µg/ml) or goat nonimmune IgG (goat-IgG; 40 µg/ml). The BrdU incorporation during the second 24 hours incubation period was determined by ELISA. The BrdU-labeling index was calculated as the ratio of the corresponding value in 10% FCS-stimulated cells. Results, given as box-and-whisker plots, were obtained from triplicate determinations in five independent experiments. The Mann-Whitney test was employed to identify differences between groups (PDGF stimulation: anti-UPA, P=0.004; goat-IgG, P>0.05 versus vehicle; bFGF stimulation: anti-UPA, P=0.004; goat-IgG, P>0.05 versus vehicle). The control group represents cells treated with 1% FCS alone (PDGF plus vehicle versus control P<0.0001; bFGF plus vehicle versus control P<0.0001).

View larger version (17K): [in a new window]   Fig. 4. Effect of polyclonal anti-UPA antibodies on S phase entry of vascular SMC stimulated with PDGF or bFGF. Growth arrested vascular SMC exposed to 20 ng/ml PDGF or 10 ng/ml bFGF in the absence or presence of anti-UPA antibodies (40 µg/ml) received 6 hour pulses of BrdU after (A) 12 hours and (B) 18 hours of stimulation. The BrdU-labeling index was calculated as described in Fig. 3. Results from five independent experiments are given as data points. Each individual point corresponds to the mean value of triplicate determinations. Points linked by lines represent data from the same experiment obtained in the absence or presence of anti-UPA antibodies. Analysis of significance for differences within pairs in each group was performed by the Wilcoxon matched-pair signed rank test (P=0.031 for each group).

View larger version (18K): [in a new window]   Fig. 5. Role of the catalytic and the growth factor domains of UPA in PDGF- or bFGF-stimulated DNA synthesis by vascular SMC. As described in Fig. 1, growth-arrested cells were exposed to PDGF or bFGF in the presence of 40 µg/ml mAb 394OA against the UPA B-chain (catalytic domain; crosshatched bars), 40 µg/ml mAb 3921 against the UPA A-chain (growth factor domain; right-hatched bars), 40 µg/ml mAb 3936 against the UPAR (inhibition UPA binding; left-hatched bars) or vehicle (open bars). Incorporation of BrdU was determined between 24 and 48 hours of stimulation with PDGF or bFGF and expressed as BrdU-labeling index. Results, given as box-and-whisker plots, are obtained from triplicate determinations in six independent experiments. P<0.001 for differences in BrdU-LI between cells treated with mAb 394OA and vehicle under stimulation with either PDGF (A) or bFGF (B).

View larger version (14K): [in a new window]   Fig. 6. Effect of p-aminobenzamidine on DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. As described in Fig. 1, growth arrested cells were exposed to PDGF () or bFGF () in the presence of 0, 62.5, 250 and 1000 µM p-aminobenzamidine for 48 hours and incubated with BrdU for the second 24 hour period. BrdU-labeling indices are presented as medians (IQR) of four independent experiments performed in triplicate. Statistical analysis was performed by the Kruskal-Wallis test (P<0.0001). The multiple comparisons' criterion was employed to identify differences between groups (P<0.001 for 250 and 1000 µM versus 0 µM in PDGF- and bFGF-stimulated cells).

View larger version (15K): [in a new window]   Fig. 7. Inhibition of PDGF- or bFGF-stimulated SMC proliferation by antibodies against UPA and p-aminobenzamidine. Cell counts [medians (IQR) of 3-4 independent experiments] were performed over periods of 7 days on PDGF- (A) or bFGF- (B) stimulated SMC cultured in the absence (hexagon) or in the presence of 40 µg/ml polyclonal goat anti-UPA 398 ([UNK]), 40 µg/ml monoclonal mouse anti-UPA 394OA (), or 250 µM of the UPA inhibitor p-aminobenzamidine ().

View larger version (16K): [in a new window]   Fig. 8. Effect of simultaneous inhibition of UPA and CK2 activities on DNA synthesis in PDGF- or bFGF-stimulated vascular SMC. Growth arrested cells were exposed to PDGF (A) or bFGF (B) in the presence of 0, 4, 6, 8 or 10 µM 4,5,6,7-tetrabromobenzotriazole (TBB) with or without the simultaneous presence of p-aminobenzamidine (p-AMB) at doses of 100 and 200 µM. Incorporation of BrdU was determined between 0 and 24 hours of stimulation with PDGF or bFGF and expressed as BrdU-labeling index. Results, given as box-and-whisker plots, are obtained from triplicate determinations in a minimum of four independent experiments. BrdU-labeling index in PDGF- or bFGF-stimulated cells was significantly reduced in all groups receiving TBB with or without p-AMB (Kruskal-Wallis test and multiple comparison's criterion for pairwise comparisons, P<0.01 for all groups receiving TBB and/or p-AMB compared with groups receiving vehicle alone). Groups treated simultaneously with TBB and p-AMB had significantly lower BrdU-LI than groups treated with TBB alone (Kruskal-Wallis test and multiple comparison's criterion for pairwise comparisons, P<0.01 for all comparisons between TBB/p-AMB-treated groups versus the corresponding TBB-treated groups unless inhibition was complete with TBB alone).

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