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Dual role for TWEAK in angiogenic regulation

Departments of Exploratory Science, Molecular Genetics and Protein Engineering, Biogen Inc., 12 Cambridge Center, Cambridge, Massachusetts 02142, USA

View larger version (14K): [in a new window]   Fig. 1. TWEAK binding to HUVECs is dose-dependent and specific. Mean fluorescence intensity (MFI) versus TWEAK concentration is shown (left), with the dotted line indicating the background MFI with the indirect detection step alone. The traces (right) correspond to (1) background fluorescence with indirect detection step alone, (2) TWEAK binding and (3) inhibition of TWEAK binding by the AB.D3 mAb.

View larger version (20K): [in a new window]   Fig. 2. Effect of TWEAK on survival of (A) HUVEC and (B) HDMEC. ECs were cultured for 48 hours in CS-C complete media (CM) or incomplete media (IM) with or without TWEAK or VEGF. Anti-TWEAK mAb AB.G11 and control Ig were added as specified on the bar graphs. Cells were stained with FITC-Annexin-V and PI, and the percentage of viable and apoptotic cells is indicated. The results with HUVEC and HDMEC are the mean percentages ±s.d. of n=4 and n=2 experiments, respectively. HUVEC cultures with TWEAK versus IM were significantly different with respect to the percentage of viable cells and the percentage of apoptotic cells (p<0.05 by Student’s t test for each comparison indicated by asterisks).

View larger version (33K): [in a new window]   Fig. 3. TWEAK enhances bFGF-dependent proliferation and migration but has no effect on VEGF-dependent proliferation and migration. HUVEC were cultured for three days in complete media (CM) or in basal media with (A) TWEAK, BBE or BBE + TWEAK, and (B) TWEAK, bFGF or combinations of these factors as indicated, and proliferation was measured by [3H]-thymidine incorporation. Data shown are the mean c.p.m. value ±s.d. of triplicate wells. Results in (A) and (B) are representative of two and four independent experiments, respectively. The proliferation in bFGF- + TWEAK-treated cultures was significantly different from that of cultures with bFGF alone or TWEAK alone (indicated by asterisks) or basal media (p values <0.05 by Student’s t test), and the difference between cultures in basal media with and without TWEAK was not significant. Blocking anti-TWEAK mAb AB.D3, nonblocking anti-TWEAK mAb BE.B3 and a control Ig Ha4/8 were also added where indicated, with results representative of two independent experiments. (C) HUVECs were cultured as described above and TWEAK, VEGF or combinations of these factors were added to basal media as indicated. Results shown are the mean c.p.m. value ±s.d. of triplicate wells and are representative of three independent experiments. The difference between VEGF and VEGF + TWEAK was not significant. (D) Confluent HUVEC monolayers treated with TWEAK, bFGF, VEGF or combinations of these factors were wounded, and repair was measured after 18 hours of culture. Results shown are the average of 6-10 experiments ±s.d. The percentage wound repair was significantly higher (Student’s t test, p<0.05) with TWEAK + bFGF compared with bFGF or TWEAK alone (indicated by asterisks) or basal media. The percentage wound repair with VEGF was significantly higher than with basal media or TWEAK but not different from that with VEGF + TWEAK.

View larger version (96K): [in a new window]   Fig. 4. TWEAK synergizes with bFGF to enhance microvessel formation. (A) Phase contrast images (all panels at 4x magnification) of HUVECs on the surface of fibrin gel matrices after three days of culture, untreated or treated with bFGF, TWEAK or bFGF + TWEAK. Results are representative of eight independent experiments. (B) Hematoxylin and eosin-stained cross-sections of the fibrin gel cultures (all panels at 20x magnification; scale bar, 100 µm). EC cells in untreated and TWEAK-treated cultures remain on the fibrin gel surface, bFGF-treated cultures exhibit invading cord-like structures, and TWEAK + bFGF-treated cultures show the structural organization of endothelial lumens (indicated by arrowheads). (C) The same cultures as in (A), with or without factors and anti-TNF and anti-IL-8 mAbs, as indicated. Capillary sprouts were counted visually using five fields per well, and the mean number per culture was normalized to the bFGF treatment, which was defined as 100%. Results are representative of seven independent experiments. The percentage increase in all of the TWEAK + bFGF treated groups was significantly higher than that in all groups treated with bFGF alone (asterisks indicate p<0.05 by Dunnett’s method), and there was no significant effect of anti-TNF, anti-IL-8 or control Ig.

View larger version (172K): [in a new window]   Fig. 5. TWEAK antagonizes the morphogenic response of ECs to VEGF. Phase contrast images (all panels at 10x magnification) of human EC on the surface of fibrin gel matrices after three days of culture, untreated or treated with VEGF, TWEAK or TWEAK + VEGF. Results are representative of 13 independent experiments.