Fig 1.
ABL increases VEGF-induced HUVEC growth and proliferation.
A) VEGF-induced EC growth monitored by the MTT assay following treatment with different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to non-stimulated cells from 3 independent experiments. B) VEGF-induced [3H]-thymidine incorporation following different doses of ABL or vehicle. The data represent the percent increase after 48 h relative to the non-stimulated cells from 3 independent experiments (n = 3). *P < 0.05 vs. vehicle, #P < 0.05 vs. VEGF.
Fig 2.
Effects of ABL on VEGF-induced HUVEC migration and tube formation.
A-B) ABL increases VEGF-induced ECs transwell migration. The data represent the number of migrated cells per microscopic field in VEGF-treated cells vs. control cells. C-D) ABL increases VEGF-induced EC transwell migration. Confluent HUVECs in a monolayer were pretreated with ABL or vehicle for 2 h and wounded with a cell scraper. After 48 h of incubation at 37°C, the number of cells that migrated across the wound edge was counted in each field. E-F) ABL increases VEGF-induced EC tube formation. The images were visualized using a phase contrast microscope (10× magnification). The total tubule length from 10 non-overlapping fields was measured by tracing the tube-like structure. The values represent the mean ± SEM from 3 independent experiments (n = 3). Scale bar: 100 μm.
Fig 3.
ABL enhances VEGF signaling in endothelial cells.
A) HUVECs were serum-starved overnight and pretreated with ABL or vehicle for 2 h, followed by exposure to VEGF-A (50 ng/mL) for various times as indicated. MAPK MAPK p44/42Thr202/Tyr204, p38Thr180/Tyr182, and AktSer473 phosphorylation in the cell lysates were measured via western blot analysis. B-D) The quantitative analysis represents the ratio of phosphorylated/total MAPK MAPK p44/42, MAPK p38, and Akt from 3 independent experiments. E) VEGFR-2Tyr1175 phosphorylation in the cell lysates was measured via western blot analysis. F) Quantitative analysis of the ratio of phosphorylated/total VEGFR-2 from three independent experiments (n = 3).
Fig 4.
ABL enhances Matrigel angiogenesis in vivo.
A) Examples of CD31 immunofluorescence staining of Matrigel plugs containing VEGF-A or control buffer. B) The quantification data represent the percent increase in CD31-positive area in comparison with the control sample (n = 6). C) Quantitative analysis of GAPDH-normalized VE-cadherin (Cdh5) mRNA expression in Matrigel plugs containing VEGF-A or control buffer that were implanted in mice (n = 3). Scale bars: 100 μm.
Fig 5.
ABL increases adult angiogenesis.
A–E) Hindlimb ischemia-induced neovascularization. Representative examples of laser-Doppler images (A) and quantification of hindlimb blood flow (B) before and after left femoral artery ligation (n = 6). Representative examples are shown. C-E) CD31 immunofluorescence staining and quantification of CD31 immunostaining of calf blood vessels (n = 6) and GAPDH-normalized VE-cadherin (Cdh5) mRNA expression in calf muscle (n = 3) 14 days after femoral artery ligation in mice. Scale bars: 100 μm.
Fig 6.
ABL inhibits VE-cadherin and VEGFR-2 complex formation in ECs.
A) Total protein extract from HUVECs pretreated with ABL or vehicle for 2 h, VEGFR-2 immunoprecipitation followed by western blot analysis of VE-cadherin. B-D) Total mRNA was purified from HUVECs pretreated with ABL or vehicle for 2 h, and VE-cadherin (Cdh5) and VEGFR-2 (Vegfr2) mRNA expression were analyzed by quantitative RT-PCR (normalized to Gapdh). ABL did not change VE-cadherin (Cdh5) and VEGFR-2 (Vegfr2) mRNA (B) or protein levels (C-D) in endothelial cells. E-F) Cell surface proteins from ABL-treated HUVECs or vehicle controls were biotinylated, captured on streptavidin Dynabeads, and subjected to Western blotting. The data obtained from 3 independent experiments (n = 3).
Fig 7.
ABL enhances VEGFR-2 internalization in ECs.
A) Colocalization of VEGFR-2 with endosome markers EEA1 in ECs. The ECs were pretreated with ABL for 2 h following fifteen minutes of VEGF stimulation, fixed, permeablized, and labeled with anti-VEGFR-2 (red) and anti-EEA1 (green) and processed for confocal microscopy. The endocytic trafficking of VEGFR2 without locating in endosome was observed in ECs (arrow). B) Colocalization of VEGFR-2 (red) and VE-cadherin (green) was observed in ECs (arrow). Nuclei were counterstained with DAPI (blue). Scale bar = 20 μm.
Fig 8.
Schematic representation of the ABL effect on VEGF signaling in ECs.
ABL inhibits VEGFR-2 and VE-cadherin association, thus promoting VEGFR-2 internalization, following by VEGF-induced VEGFR-2 phosphorylation and downstream signaling.